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.

Combining radioimmunotherapy with antihypoxia therapy 2-deoxy-D-glucose results in reduction of therapeutic efficacy.

PURPOSE: The efficacy of solid tumor radioimmunotherapy is reduced by heterogeneous tumor distribution of the radionuclide, with dose mainly deposited in the normoxic region and by the relative radioresistance of hypoxic tumor cells. In an attempt to overcome these challenges, radioimmunotherapy was combined with 2-deoxy-d-glucose (2DG), a hypoxia-selective cytotoxic inhibitor of glucose metabolism. EXPERIMENTAL DESIGN: In vitro toxicity of 2DG in LS174T cultures was tested using a colony-forming assay. The effect of combining 2DG with radioimmunotherapy in vivo was tested by administering radiolabeled anti-carcinoembryonic antigen antibody ([(131)I]A5B7 IgG1 whole monoclonal) to nude mice bearing s.c. LS174T tumors, followed by 10 daily injections of 2DG (2.0 g/kg). Tumors were measured to assess therapeutic efficacy. RESULTS: Data from in vitro studies confirmed 2DG cytotoxicity in this cell line. Greater toxicity was observed under standard laboratory conditions and in hypoxic cultures than at intermediate, physiologically relevant levels of glucose and oxygen. Alone, 2DG had no effect on in vivo tumor growth (P = 0.377 compared with saline-treated controls). Combination of radioimmunotherapy with 2DG reduced the therapeutic effect of radioimmunotherapy (e.g., 150 microCi (131)I alone mean survival time, 48.33 +/- 16.83 days; combined with 2DG, 30.67 +/- 5.62 days, P = 0.038). CONCLUSIONS: The combination investigated had a detrimental effect on survival. It is suggested that a cellular metabolic response to more aggressive therapy, previously reported in vitro, caused this. The results of this study have implications for the clinical application of combined cancer therapies with an antimetabolic modality component.

Combretastatin A-4-phosphate effectively increases tumor retention of the therapeutic antibody, 131I-A5B7, even at doses that are sub-optimal for vascular shut-down.

Radioimmunotherapy using 131I-A5B7, an anti-CEA antibody, in combination with the vascular disrupting agent, combretastatin A4-phosphate (CA-4-P, 200 mg/kg), has produced tumor cures in SW1222 colorectal xenografts. CA-4-P causes acute tumor blood vessel shutdown, which can be monitored in clinical trials using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). The purpose of this study was to determine the magnitude of the anti-vascular effect of CA-4-P in the SW1222 tumor, at 200 mg/kg and at lower, more clinically relevant doses, using conventional assays; relate effects to changes in DCE-MRI parameters and determine the corresponding effects on tumor retention of 131I-A5B7. The tumor vascular effects of 30, 100 and 200 mg/kg CA-4-P were determined, at 4- and 24-h post-treatment, using DCE-MRI, uptake of Hoechst 33342 for tumor vascular volume and conventional histology for necrosis. The effect of CA-4-P on tumor and normal tissue 131I-A5B7 retention was also determined. A significant reduction in tumor DCE-MRI kinetic parameters, the initial area under the contrast agent concentration time curve (IAUGC) and the transfer constant (Ktrans), was demonstrated at 4 h after CA-4-P, for all dose levels. These effects persisted for at least 24 h for the 200 mg/kg group but not for lower doses. A similar pattern was seen for vascular volume and necrosis. Despite this dose response, all three dose levels increased tumor retention of radio labeled antibody to a similar degree. These results demonstrate that moderate tumor blood flow reduction following antibody administration is sufficient to improve tumor antibody retention. This is encouraging for the combination of CA-4-P and 131I-A5B7 in clinical trials.

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.

Adrenomedullin haploinsufficiency predisposes to secondary lymphedema.

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor-like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (Adm(AM+/Δ) animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both Adm(AM+/+) and Adm(AM+/Δ) mice. However, only Adm(AM+/Δ) animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in Adm(AM+/Δ) mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

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.

Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy.

MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.

Transformation of human mesenchymal stem cells increases their dependency on oxidative phosphorylation for energy production.

An increased dependency on glycolysis for ATP production is considered to be a hallmark of tumor cells. Whether this increase in glycolytic activity is due mainly to inherent metabolic alterations or to the hypoxic microenvironment remains controversial. Here we have transformed human adult mesenchymal stem cells (MSC) using genetic alterations as described for differentiated cells. Our data suggest that MSC require disruption of the same pathways as have been shown for differentiated cells to confer a fully transformed phenotype. Furthermore, we found that MSC are more glycolytic than primary human fibroblasts and, in contrast to differentiated cells, do not depend on increased aerobic glycolysis for ATP production during transformation. These data indicate that aerobic glycolysis (the Warburg effect) is not an intrinsic component of the transformation of adult stem cells, and that oncogenic adaptation to bioenergetic requirements, in some circumstances, may also rely on increases in oxidative phosphorylation. We did find, however, a reversible increase in the transcription of glycolytic enzymes in tumors generated by transformed MSC, indicating this is a secondary phenomenon resulting from adaptation of the tumor to its microenvironment.

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.