R Boden

Polyethylene glycol modification of a galactosylated streptavidin clearing agent: effects on immunogenicity and clearance of a biotinylated anti-tumour antibody.

Effective radioimmunotherapy is limited by slow antibody clearance from the circulation, which results in low tumour to blood ratios and restricts the dose of radiolabelled anti-tumour antibody that can be safely administrated. Avidin and streptavidin clearing agents have been shown to effectively complex and clear radioactive biotinylated antibodies from the circulation, but their immunogenicity may limit their repeated use. We have investigated whether polyethylene glycol (PEG) modification can reduce the immunogenicity of our galactosylated streptavidin (gal-streptavidin) clearing agent without altering its effectiveness as a clearing agent. The immune response evoked in mice after intraperitoneal infection of 30 micrograms of gal-streptavidin was decreased after PEG modification, as shown by lower antibody titres and a reduction in the number of mice that elicited an anti-gal-streptavidin response. The effect of PEG-modified gal-streptavidin on the blood clearance and tumour localisation of a 125I-labelled biotinylated anti-CEA was investigated in the LS174T human colon carcinoma xenograft in nude mice. Although PEG modified gal-streptavidin bound the [125I]biotinylated antibody in vivo, effective clearance from the circulation was inhibited, resulting in very little reduction in the levels of circulation radioactivity, together with a decrease in the antibody localised to the tumour.

A Mouse Model for Calculating the Absorbed Beta-Particle Dose from 131 I- and 90 Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor

Abstract Flynn, A. A., Green, A. J., Pedley, R. B., Boxer, G. M., Boden, R. and Begent, R. H. J. A Mouse Model for Calculating the Absorbed Beta-Particle Dose from 131I- and 90Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor. Radiat. Res. 156, 28–35 (2001). Conventional internal radiation dosimetry methods assume that the β-particle energy is absorbed uniformly and completely in the source organ and that the radioactivity is distributed uniformly in the source. However, in mice, a considerable proportion of the β-particle energy can escape the source organ, resulting in large cross-organ doses. Furthermore, the distribution of radioactivity is generally heterogeneous in kidney and tumor. Therefore, a model was developed to account for cross-organ doses and for the effects of heterogeneity in kidney and tumor in mice for two of the most important radionuclides used in therapy, 131I and 90Y. Most mouse organs were modeled as single-compartment ellipsoids or cylinders, while heterogeneity in kidney and in tumor was addressed by using two compartments to represent the cortex and the medulla and viable and necrotic cells, respectively. The dimensions of these models were taken from previous studies, with the exception of kidney and tumor, which were defined using radioluminography and mosaics of high-power microscopy images. The absorbed fractions in each compartment were calculated using β-particle point dose kernels. The self-organ dose was significantly higher for 131I compared to 90Y in all compartments, but a considerable amount of β-particle energy was shown to escape the source organ for both radionuclides, with as much as 85% and 36% escaping the marrow for 90Y and 131I, respectively. The cortex was found to occupy a greater proportion of the total kidney volume than the medulla, and consequently the self-dose was higher in the cortex. In addition, the thickness of the viable shell in the tumor increased with tumor size, as did the self-dose fractions in both necrotic and viable areas. This dosimetry model improves dose estimates in mice and gives a conceptual basis for considering dosimetry in humans.

Clearance of circulating radio-antibodies using streptavidin or second antibodies in a xenograft model.

The improved tumour to non-tumour ratios needed for effective tumour targeting with antibodies requires that blood background radioactivity is reduced. We investigated the effect of streptavidin as a clearing agent for 125I-labelled biotinylated anti-CEA antibodies in a human colon carcinoma xenograft model. By comparing the biodistribution of the monoclonal antibody A5B7 with four, nine or 22 biotins per antibody molecule, we investigated how the degree of biotinylation of the primary radiolabelled antibody affects its clearance with streptavidin. Limiting the degree of biotinylation limited blood clearance, whereas nine or 22 biotins per antibody molecule resulted in a 13- to 14-fold reduction in blood radioactivity, the streptavidin-biotinylated antibody complexes clearing rapidly via the liver and spleen. Although a reduction in tumour activity was also seen, a 6.6-fold improvement in the tumour to blood ratio was achieved. A comparative study of streptavidin versus second antibody clearance was carried out using the polyclonal antibody PK4S biotinylated with 12 biotins per antibody molecule. This study indicated that second antibody was superior for clearance of the polyclonal antibody, resulting in a larger and faster reduction in blood radioactivity and improved tumour to blood ratios. In this case the primary antibody was polyclonal, and therefore non-uniformity of biotinylation may affect complexation with streptavidin. Therefore, the degree of biotinylation and type of antibody must be carefully considered before the use of streptavidin clearance.

Antibody and radionuclide characteristics and the enhancement of the effectiveness of radioimmunotherapy by selective dose delivery to radiosensitive areas of tumour.

Purpose : Estimating the absorbed dose to tumour relative to normal tissues has often been used in the assessment of the therapeutic efficacy of radiolabelled antibodies for radioimmunotherapy. Typically, the calculations assume a uniform dose deposition and response throughout the tumour. However, the heterogeneity of the dose delivery and response within tumours can lead to a radiobiological effect inconsistent with dose estimates. The aim was to assess the influence of antibody and radionuclide characteristics on the heterogeneity of dose deposition. Materials and methods : Quantitative images of the temporal and spatial heterogeneity of a range of antibodies in tumour were acquired using radioluminography. Subsequent registration with images of tumour morphology then allowed the delineation of viable and necrotic areas of tumour and the measurement of the antibody concentration in each area. A tumour dosimetry model then estimated the absorbed dose from 131 I and 90 Y in each area. Results : Tumour-specific antibodies initially localized in the viable radiosensitive areas of tumour and then penetrated further into tumour with continued tumour accretion. Multivalent antibodies were retained longer and at higher concentrations in viable areas, while monovalent antibodies had greater mobility. In contrast, non-specific antibodies penetrated into necrotic regions regardless of their size. As a result, multivalent, specific antibodies delivered a significantly larger dose to viable cells compared with monovalent antibodies, while non-specific antibodies deposited most of the dose in necrotic areas. There was a significant difference in dose estimates when assuming a unifrom dose deposition and accounting for heterogeneity. The dose to the viable and necrotic areas also depended on the properties of the radionuclide where antibodies labelled with 131 I generally delivered a higher dose throughout the tumour even though the instantaneous dose-rate distribution for 90 Y was more uniform. Conclusions : The extent of heterogeneity of dose deposition in tumour is highly dependent on the antibody characteristics and radionuclide properties, and can enhance therapeutic efficacy through the selective dose delivery to the radiosensitive areas of tumour.

Effectiveness of radiolabelled antibodies for radio-immunotherapy in a colorectal xenograft model: a comparative study using the linear--quadratic formulation.

Purpose : To develop a model that relates the pattern of dose delivery during radio-immunotherapy to biological effect. This model was used to assess the efficacy of a range of antibodies labelled with 131 I, 186 Re and 90 Y. Materials and methods : Pharmacokinetic data were obtained by injecting tumour-bearing nude mice with radiolabelled antibody. The dose-rate in bone marrow and tumour was then given by a two-compartment model description of the pharmacokinetics combined with the radionuclide properties. Response characteristics of tumour and marrow were defined in terms of radiosensitivity, repair capacity and proliferation, and the biological effect was assessed using the linear-quadratic formulation. Results : Tumour-specific antibodies with intermediate molecular weight and clearance from the circulation delivered the most effective doses to tumour due to their rapid uptake and prolonged retention in tumour coupled with efficient clearance from blood. Matching the radionuclide with antibody pharmacokinetics and tumour type further increased this effect. Conclusions : The model improves conceptual understanding of the relationship of parameters affecting therapy and makes it possible to optimize radio-immunotherapy by selecting the most effective antibody and radionuclide according to tumour biology.PURPOSE To develop a model that relates the pattern of dose delivery during radio-immunotherapy to biological effect. This model was used to assess the efficacy of a range of antibodies labelled with 131I, 186Re and 90Y. MATERIALS AND METHODS Pharmacokinetic data were obtained by injecting tumour-bearing nude mice with radiolabelled antibody. The dose-rate in bone marrow and tumour was then given by a two-compartment model description of the pharmacokinetics combined with the radionuclide properties. Response characteristics of tumour and marrow were defined in terms of radiosensitivity, repair capacity and proliferation, and the biological effect was assessed using the linear quadratic formulation. RESULTS Tumour-specific antibodies with intermediate molecular weight and clearance from the circulation delivered the most effective doses to tumour due to their rapid uptake and prolonged retention in tumour coupled with efficient clearance from blood. Matching the radionuclide with antibody pharmacokinetics and tumour type further increased this effect. CONCLUSIONS The model improves conceptual understanding of the relationship of parameters affecting therapy and makes it possible to optimize radio-immunotherapy by selecting the most effective antibody and radionuclide according to tumour biology.

The effect of serum CEA on the distribution and clearance of anti-CEA antibody in a pancreatic tumour xenograft model.

A human pancreatic adenocarcinoma was used to develop two histologically distinct xenograft lines, one associated with high levels (180-2000 ng ml-1) and one with low levels (greater than 2.0 less than 8.0 ng ml-1) of serum carcinoembryonic antigen (CEA). A strong correlation was found between tumour size and both circulating and tumour CEA levels in the former group, and also correlation at the 5% level between tumour size and serum CEA in the latter. Administration of either monoclonal or polyclonal 125I-anti-CEA antibody led to the formation of intravascular antigen-antibody immune complexes in mice with high CEA levels, and these were rapidly cleared by the liver, deiodination commencing within the first hour. Blood activity was reduced to 20% of the injected dose by 15 min, and by 24 h the radioactivity in all tissues except muscle was significantly below that found in either the low CEA group or in mice without tumours. No difference in radio-antibody clearance pattern was found between mice without tumours and the group with low levels of serum CEA. In spite of higher levels of CEA within the tumour in mice with elevated serum CEA, the rapid clearance of antigen-antibody complexes reduced tumour localisation to one quarter of that seen in mice with low serum, and correspondingly low tumour, CEA levels. Gamma-camera imaging confirmed these results. Possible implications to patient selection and treatment are discussed.

Higher dose and dose-rate in smaller tumors result in improved tumor control

Small tumors are more sensitive to radioimmunotherapy (RIT) than larger ones. A greater proportion of viable radiosensitive areas in small tumors, higher antibody uptake, and radiation dose may be responsible. Six groups of mice with small (median tumor size 0.06 cm3) or large LoVo xenografts (median tumor size 0.38 cm3) received either RIT using a 131I-labeled anti-CEA antibody A5B7, 5-fluorouracil (5-FU) modulated with folinic acid (FA), or no treatment. The % injected activity/gram, antibody distribution in viable and necrotic areas, and dose distribution were determined. High-power microscopy images of the original section were reconstructed to estimate the proportion of viable areas. Mice with small and large tumors grew significantly less rapidly when treated with RIT compared to the control group (p<0.0004 and p<0.003, respectively), while 5-FU was ineffective. Small tumors treated with RIT grew less than large tumors (p<0.02). A higher amount of % injected activity/gram of tumor (median 26.6% vs. 8.1%,p=0.0007) and a higher dose-rate were found in small tumors at 24 hours post injection (viable areas: 56.2±23.7 vs. 13.3±7 cGy/h, necrosis 19.2±16.3 vs. 4.9±4.7 cGy/h,p=0.0007). It appears that as viable tumor masses grow the access to them decreases and this has a fourfold effect on dose delivered for RIT in this example. These data support the consideration of use of RIT for adjuvant treatment in colon cancer.

Clearance of yttrium-90-labelled anti-tumour antibodies with antibodies raised against the 12N4 DOTA macrocycle.

Radioimmunotherapy (RIT) is currently limited by toxicity to normal tissues as a result of prolonged circulating radioantibody in the blood. In this study, the use of a clearing antibody was investigated (second antibody) in an attempt to reduce blood background levels of [90Y]A5B7 immunoglobulin G (IgG) activity, and, therefore, improve the therapeutic tumour-blood ratio in nude mice bearing human colorectal tumour xenografts. The second antibody was raised against the 12N4 macrocycle group used for chelation of 90Y, and is, thus, applicable to any anti-tumour antibody labelled with this methodology. Second antibody was administered 18, 24 or 48 h after radiolabelled antibody injection and produced up to a tenfold reduction in blood levels and a tenfold improvement in tumour-blood ratios. This has the advantage of reducing the risk of myelotoxicity caused by prolonged retention of activity in the blood. For all normal tissues, there was a similar or slightly lower uptake of [90Y]IgG with second antibody clearance, apart from a transient rise in liver activity due to complexes of primary and secondary antibody clearing via the liver. As a result of clearance of [90Y]IgG from the blood pool, there was an associated fall in the amount of antibody at the tumour site (up to 3.3-fold) at later time points for mice injected with second antibody. However, despite this, tumour-blood ratios remained superior to the control group at these later time points. Estimated dosimetry evaluation revealed that total dose to normal tissues, blood and tumour was lower than for the non-clearance group. Surprisingly, however, there was little improvement in total estimated tumour-blood dose ratio over the time period studied. This was probably because the majority of the dose was delivered to both the blood and tumour within the first 24 h after administration of [90Y]IgG, so that giving the clearing agent after this time did not produce a large difference in total estimated dose. The anti-macrocycle second antibody proved to be a successful clearing agent and could potentially be applied to any anti-tumour antibody coupled with the 12N4 macrocycle. In the light of the estimated dosimetry results described here, it would probably be most useful given at earlier time points (i.e. before 18 h after injection of primary antibody) to produce an improved tumour-blood ratio of total dose. Development of this strategy may allow higher levels of activity to be administered for RIT, and repeated dosing regimens.

Spatial accuracy of 3D reconstructed radioluminographs of serial tissue sections and resultant absorbed dose estimates.

Many agents using tumour-associated characteristics are deposited heterogeneously within tumour tissue. Consequently, tumour heterogeneity should be addressed when obtaining information on tumour biology or relating absorbed radiation dose to biological effect. We present a technique that enables radioluminographs of serial tumour sections to be reconstructed using automated computerized techniques, resulting in a three-dimensional map of the dose-rate distribution of a radiolabelled antibody. The purpose of this study is to assess the reconstruction accuracy. Furthermore, we estimate the potential error resulting from registration misalignment, for a range of beta-emitting radionuclides. We compare the actual dose-rate distribution with that obtained from the same activity distribution but with manually defined translational and rotational shifts. As expected, the error produced with the short-range 14C is much larger than that for the longer range 90Y; similarly values for the medium range 131I are between the two. Thus, the impact of registration inaccuracies is greater for short-range sources.