Radioimmunotherapy with alpha-particle-emitting immunoconjugates.

Alpha particles are energetic short-range ions whose higher linear energy transfer produces extreme cytotoxicity. An alpha-particle-emitting radioimmunoconjugate consisting of a bismuth-212-labeled monoclonal immunoglobulin M specific for the murine T cell/neuroectodermal surface antigen Thy 1.2 was prepared. Analysis in vitro showed that the radioimmunoconjugate was selectively cytotoxic to a Thy 1.2+ EL-4 murine tumor cell line. Approximately three bismuth-212-labeled immunoconjugates per target cell reduced the uptake of [3H]thymidine by the EL-4 target cells to background levels. Mice inoculated intraperitoneally with EL-4 cells were cured of their ascites after intraperitoneal injection of 150 microcuries of the antigen-specific radioimmunoconjugate, suggesting a possible role for such conjugates in intracavitary cancer therapy.

Modeling of dose to tumor and normal tissue from intraperitoneal radioimmunotherapy with alpha and beta emitters.

Dose distributions for normal and tumor tissues from intraperitoneally administered radiolabeled antibodies have been calculated for 90-Yttrium (90Y), 131-Iodine (131I), and 211-Astatine (211At). The dose calculations use data on the activity of intraperitoneal fluid administered, the percent injected dose/gm uptake by tumor, biological half life, and a model for diffusion of antibody/radionuclide complex into peritoneal tissues. Calculations are performed for planar and hemispherical tumor shapes, ranging in size to establish the influence of geometry on dose distribution. Calculations for tumor geometry obtained from biopsies are also performed. When the activity is concentrated on or near the tumor surface, the maximum dose to a planar tumor for a 20 mci administration of 90Y is approximately 60 Gy, and falls rapidly to 50% of this value within 1 mm. However, for a hemispherical tumor, the dose is a maximum of 26 Gy, with an average of approximately 20 Gy. The surface dose from 131I (130 mci) is 240 Gy, and diminishes to 20 Gy in .05 cm in the planar case, whereas a hemispherical tumor receives a dose of 90 Gy over a large fraction of the volume, with the distal portions receiving 40 Gy. The surface dose for an administration of 70 mci of 211 At is 450 Gy and decreases to 50% of this value in 30 microns. Both surface geometry and tumor size are important determinants in the heterogeneity of tumor dose, as are the dose administered, antibody uptake, biodistribution, and residence time factors. These initial studies suggest that the size of disease which may be effectively treated is much less than the range of the particle emitted by radiolabeled antibodies. Furthermore, therapy is ultimately limited by the degree to which the antibody/radionuclide complex can diffuse and permeate the tumor.

Alpha particle radio-immunotherapy: animal models and clinical prospects.

Short-lived isotopes that emit alpha particles have a number of physical characteristics which make them attractive candidates for radioimmunotherapy. Among these characteristics are high linear energy transfer and correspondingly high cytotoxicity; particle range limited to several cell diameters from the parent atom; low potential for repair of alpha-induced DNA damage; and low dependence on dose rate and oxygen enhancement effects. This report reviews the synthesis, testing and use in animal models of an alpha particle emitting radioimmunoconjugate constructed via the noncovalent chelation of Bismuth-212 to a monoclonal IgM antibody specific for the murine T cells/neuroectodermal surface antigen, Thy 1.2. These 212Bi-anti-Thy 1.2 immunoconjugates are capable of extraordinary cytotoxicity in vitro, requiring approximately three 212Bi-labeled conjugates per target cell to suppress 3H-thymidine incorporation to background levels. The antigen specificity afforded by the monoclonal antibody contributes a factor of approximately 40 to the radiotoxicity of the immunoconjugate. Animals inoculated with a Thy 1.2+ malignant ascites were cured of their tumor in an antigen-specific fashion by intraperitoneal doses of approximately 200 microCi per mouse. Alpha particle emitting radioimmunoconjugates show great potential for regional and intracavitary molecular radiotherapy.

211At radiocolloid therapy: further observations and comparison with radiocolloids of 32P, 165Dy, and 90Y.

We compared the therapeutic efficacy of alpha and beta emitting radiocolloids for the treatment of experimental malignant ascites. 211At is an almost pure alpha-emitter. As 211At-tellurium colloid, the dose survival curve is linear and extrapolates through the origin in a manner similar to other high linear energy transfer radiations. Doses of 25 microCi were curative. Less than curative doses showed a graded prolongation of median survival. In cured mice, long term histological changes were seen in thyroid tissue. Acute changes were seen in the gastrointestinal tract as early as 2 hr after radiocolloid administration; these changes reached a plateau at 6 hr and were essentially gone 36 hr later. By comparison, radiocolloids of the beta emitters 32P, 165Dy and 90Y were not curative, but relatively large doses did substantially prolong median survival. The doses for maximal effect were 150 microCi 32P-chromic phosphate, 8000 microCi ++165Dy-ferric hydroxide macroaggregates and 200 microCi 90Y-citrate. The most compelling reason for the increased therapeutic efficacy of 211At-tellurium colloid is the direct and densely ionizing character of the emitted alpha radiations.

Iodine-125-NRLU-10 kinetic studies and bismuth-212-NRLU-10 toxicity in LS174T multicell spheroids.

Alpha emitter-labeled antibodies (Abs) are of considerable interest in cancer therapy. Alpha particles are densely ionizing and therefore have a high radiobiologic effectiveness, and the cell killing produced is influenced very little by dose rate or hypoxic conditions. LS174T human colon adenocarcinoma spheroids were used in this study to evaluate the efficacy of alpha emitter-labeled Abs in a three-dimensional model. NRLU-10, an IgG2b Ab to a pancarcinoma antigen, and its Fab fragment were used. Initial kinetic studies using 125I-NRLU-10 revealed that a large number of binding sites/cell and high Ab affinity led to slow Ab penetration. This effect could be overcome by increasing the Ab concentration ten-fold for Fab but not for intact Ab. Bismuth-212-NRLU-10 therapy was very effective in killing single cells (over 3 log reduction in surviving fraction) but was ineffective in spheroids (less than 1 log reduction). This was likely due to inadequate penetration into the spheroids before the 212Bi decayed. The use of higher Ab concentrations, tumors with fewer antigenic sites/cell for the Ab being used, lower affinity Abs, alpha emitters with longer half-lives, and pretargeting with bifunctional Ab are all potential ways of increasing the efficacy of alpha emitter-labeled Abs for cancer therapy.

Manganese-52m, a new short-lived, generator-produced radionuclide: a potential tracer for positron tomography.

A new generator system has been developed using the Fe-52 leads to Mn-52m parent-daughter pair. Fe-52, half-life 8.3 hr, is isolated on an anion-exchange column, and Mn-52m is eluted in hydrochloric acid. Breakthrough is less than 0.01% and the yield is 75%. The 21.1-min half life of Mn-52m is ideal for use in sequential studies, but is long enough to permit radiochemical manipulations to control biodistribution. Animal studies indicate that Mn-52m is an ideal nuclide for myocardial imaging, combining rapid blood clearance and high concentration in the myocardium. An added advantage is that Mn-52m decays 98% by positron emission and is useful for positron computer tomography.

Localization of pulmonary human sarcoma xenografts in athymic nude mice with indium-111-labeled monoclonal antibodies.

In order to study localization of metastatic tumors with a radiolabeled monoclonal antibody, a pulmonary metastases model was devised in athymic mice. Metastatic pulmonary sarcoma colonies were verified by histological examination. A murine monoclonal antibody (MAb 19-24) directed against a human sarcoma antigen was labeled with indium-111 (111In) by use of the linker 1-(p-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bz-DTPA). MAb P3 was similarly labeled as a negative control. In the group given MAb 19-24, the percent injected dose per gram lung tissue bearing tumor colonies (30.1%, 29.6%, and 27.7% on Days 1, 2, and 3, respectively) was significantly (p less than 0.05) higher than in those receiving MAb P3. Hepatic activities of both 111In-MAb 19-24 and 111In-MAb P3 were low. The lungs with tumor colonies demonstrated clearest images on Day 3. The specific binding of 111In-SCN-Bz-DTPA-labeled MAb 19-24 to pulmonary xenografts without appreciable liver uptake indicates that it may be useful in the clinical localization of pulmonic metastatic lesions.

New method for the chelation of indium-111 to monoclonal antibodies: biodistribution and imaging of athymic mice bearing human colon carcinoma xenografts.

B72.3, a murine monoclonal antibody (MAb) that reacts with 85% of human colon carcinomas as well as other epithelial neoplasias, was labeled with 111In using four chelating agents: 1-(p-isothiocyanatobenzyl)-DTPA (SCN-Bz-DTPA), isobutylcarboxycarbonic anhydride (MA-DTPA), cyclic anhydride (CA-DTPA), and 1-(p-isothiocyanatobenzyl)-ethylenediaminetetraacetic acid (SCN-Bz-EDTA). Comparative biodistribution and imaging studies were performed in athymic mice bearing human colon carcinoma xenografts (LS-174T). Tumor uptake of radiolabel was very similar between the chelates (30% ID/g) and tumors were identified in scintigraphic images with all the chelate-antibody complexes. The uptake by normal organs, especially the liver, was greater for MA-DTPA, CA-DTPA, and SCN-Bz-EDTA chelate-B72.3 IgG (1.3:1 to 2.5:1) in comparison to that found with the B72.3-SCN-Bz-DTPA (approximately 5:1) and abdominal organ, and uptake was very prominent on imaging with these chelate-MAb complexes but was virtually absent in the mice injected with B72.3-SCN-Bz-DTPA. Purification of the MAb-chelate complex by Sephadex G-50 chromatography followed by HPLC using a TSK-3000 column provided better subsequent biodistribution and also resulted in clearer images as compared to MAb chelate complexes purified by less rigorous purification protocols. We conclude that the 111In-SCN-Bz-DTPA complex is superior, at least when bound to MAb B72.3, to other chelate-complexes currently in use.

Improved sarcoma imaging and reduced hepatic activity with indium-111-SCN-Bz-DTPA linked to MoAb 19-24.

A murine monoclonal antibody (MoAb 19-24) directed against a human sarcoma antigen was prepared and labeled with 111In by use of the linker 1-(p-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bz-DTPA). Imaging and biodistribution of this radioimmunocomplex were evaluated. MoAb P3 was similarly labeled as a negative control. Of 24 athymic mice bearing s.c. human fibrosarcoma, 12 received 10 microCi of [111In]MoAb 19-24 and 12 received 10 microCi of [111In]MoAb P3. The mice were imaged at 24, 48, 68, or 168 hr, after i.p. injection. Region of interest and biodistribution analysis showed maximum localization of MoAb 19-24 in the tumor at 72 hr with maximum liver localization of 24 hr. Analysis of MoAb P3 showed maximum tumor and liver activity at 24 hr. Tumor specificity studies were also conducted in three nude mice bearing a sarcoma in the left flank and a Burkitt's lymphoma in the right flank. Selective uptake was seen in the sarcoma but not in the lymphoma. The excellent uptake of [111In]SCN-Bz-DTPA-MoAb 19-24 in sarcoma without appreciable liver activity indicates that it may be a useful tumor imaging agent in man.

Halogenated pyrimidines as radiosensitizers for high-LET radiation.

The incorporation of iododeoxyuridine (IdUrd) into Chinese hamster cells was examined as a possible radiosensitizer for fission spectrum neutrons. Dose-response curves comparing both X rays and neutrons in the same cell line with the same IdUrd replacement showed a similar radiation enhancement for IdUrd incorporation. Enhancement ratios at the 1% survival level were 1.8 for X rays and 1.5 for fission spectrum neutrons. While the mechanism of this enhancement in the response for fission neutron radiation is unclear, these positive data should support further exploration to determine if halogenated pyrimidine incorporation results in sensitization for neutron energies employed in therapy.

Morphological, biochemical, and molecular changes in endothelial cells after alpha-particle irradiation.

The response of cultured bovine aortic endothelial (BAE) cells after exposure to alpha-particle radiation from chelated 212Bi has been evaluated. The results suggest that even relatively high doses of alpha-particle radiation from 212Bi (20-72 Gy) cause only minor acute changes in the morphology of BAE cells (light and electron microscopy) under conditions of confluent monolayer growth. Significant morphological changes can be detected in cells that detach from the monolayer, though it is unclear whether these changes represent a genuine response to irradiation or reflect the causes or effects of monolayer detachment with the consequent loss of intercellular biochemical communication. After alpha-particle irradiation (20-40 Gy) angiotensin-converting-enzyme activity was not detectable in the monolayer culture medium but was significantly decreased within the cell monolayer. Neutral-elution-assay data demonstrated that DNA double-strand-break (DSB) damage occurred in these cells and that about 35% of the DSBs were repairable.

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

Cellular kinetics, dosimetry, and radiobiology of alpha-particle radioimmunotherapy: induction of apoptosis.

Though clinical results for radioimmunoconjugate therapy of most common epithelial tumors have been disappointing, dramatic responses have been observed repeatedly in the treatment of high- and low-grade malignant lymphomas. This high clinical responsiveness after radioimmunoconjugate therapy sometimes appears to be out of proportion to the calculated radiation dose absorbed by the lymphoma tissue. Here we describe some key aspects of the kinetics, dosimetry, and cellular radiobiology of murine lymphoma cells exposed to 212Bi-radiolabeled alpha-particle-emitting immunoconjugates specific for the differentiation antigen Thy 1.2. Approximately 25 cell-bound alpha-particle-emitting immunoconjugates per target cell were required to reduce clonogenic survival by 90% (the radiobiological D10). Serial kinetic analyses of the antibody and radioisotope components of the immunoconjugates revealed significant levels of dechelation and up to 7.5% cellular internalization of the isotope. Cellular radiation dosimetry performed by Monte Carlo computer simulation of alpha-particle energy deposition patterns based on the observed radiopharmacokinetics showed that the D10 resulted from approximately four alpha-particle traversals through the nucleus, corresponding to an absorbed radiation dose of approximately 0.95 Gy to the cell nucleus. Electron micrographs and DNA gel studies of murine lymphoma cells undergoing radioimmunoconjugate therapy in vivo and in vitro demonstrated bizarre blebbing patterns, condensation of chromosomal material, and internucleosomal DNA fragmentation patterns characteristic of programmed cell death (apoptosis). We conjecture that the efficacy of radioimmunoconjugates against responsive cell types may be the result of passive DNA damage by ionizing radiation and the initiation of apoptosis in response to radioimmunotherapy.

In vitro exposure of mammalian cells to radon: dosimetric considerations.

We have developed a model to calculate the dose to the cell nucleus in cells exposed in suspension to radon and/or radon progeny. The model addresses the influence of (1) different radiation qualities and energies in the irradiation milieu; (2) the contribution to dose from radioactivity in the medium surrounding the cell after exposure to the radon gas as well as that from excess radon progeny associated with the cell; (3) the geometry of the cell and of the radiosensitive target, the cell nucleus; (4) the intracellular localization of the radionuclides; (5) attenuation of the alpha particles by the cytoplasm; (6) the radionuclide concentrations in the medium; and (7) the length of exposure. Investigation of the influence of these various parameters was made using an irradiation system in which cells were exposed to 212Bi, which decays to stability with the emission of an alpha particle (either 6.05 or 8.78 MeV). The information from these studies was then used to develop the system further for more complex systems in which 222Rn and its progeny are present. The model takes into account the contribution of dose from different radiation sources using scintillation counts of the medium and the cells, and it is useful for calculations of dose in situations where cells are exposed in suspension culture.

Accumulation of indium-111-labeled human low density lipoprotein in the rabbit aorta: Implications for nuclear imaging of vascular lesions.

Nuclear imaging of atheromata must distinguish lesions from both blood pool and normal arterial tissue. We have examined spatial and temporal variations of indium-111-labeled human low density lipoprotein (LDL) accumulation in rabbit aortas. LDL-derived In-111 activity was time-independent in lesion-resistant regions of aortas from normal and hypercholesterolemic animals (mean 2.9 × 10(-6) percent injected activity per milligram tissue [%IA/mg]) and in lesion-prone regions of normal aortas (mean 7.1 × 10(-6) %IA/mg). In contrast, activity in sudanophilic lesions of hypercholesterolemic rabbit aortas reached a peak of 31 × 10(-6) %IA/mg at 92 hours postinjection. The mean ratio between activity in lesions versus lesion-resistant regions described a broad convex curve with minima of 4:1 at 14 hours and 136 hours and a peak of 14:1 measured at 72 hours postinjection. The mean ratio between In-111 in lesions and blood followed a sigmoid curve, rising exponentially from 1:25 at 14 hours to 1:3 by 72 hours postinjection. We conclude that optimal signal-to-noise ratios for monitoring atheroma-associated LDL-derived radioactivity occur late, not before about 3 days postinjection. Therefore, LDL labeled with In-111 or even longer-lived radionuclides holds the greatest promise for effective clinical nuclear imaging of atherosclerosis.

Comparison of radon-daughter-induced effects in repair-proficient and repair-deficient CHO cell lines.

The radiobiological effects of the radon daughter 212Bi were investigated in the Chinese hamster ovary cell line AA8 and its radiosensitive derivative EM9. EM9 cells rejoin radiation-induced DNA strand breaks more slowly than do AA8 cells. Three endpoints were examined: cell killing, G2-induced chromosome aberration frequency, and mutation induction at the hypoxanthine (guanine) phosphoribosyltransferase (HGPRT) locus. Cells were exposed to the alpha-emitter 212Bi chelated to diethylenetriaminepentaacetic acid (212Bi-DTPA). As expected, 212Bi-DTPA was more effective than X-rays in producing cytotoxicity, chromosome aberrations, and gene mutations. The relative biological effectiveness (RBE) for all three endpoints ranged from about 2 for chromosome aberrations to 4.4 for mutation induction. EM9 was more sensitive than AA8 cells to the cytotoxic and clastogenic effects of both X-rays and 212Bi-DTPA, suggesting that the repair deficiency in EM9 cells affects response to low- and high-linear energy transfer (LET) radiation for these endpoints. There was no significant difference between these two cell lines in their mutagenic response to X-rays and AA8 was slightly more sensitive to the mutagenic effects of alpha radiation. These results suggest that alterations in DNA repair ability may affect response of cells to both low- and high-LET radiation-induced cytotoxicity and clastogenicity, but they appear to have little effect on gene mutation induction.

G2M arrest and apoptosis in murine T lymphoma cells following exposure to 212Bi alpha particle irradiation.

Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET alpha-radiation from 212Bi-DTPA or to gamma-radiation from a 137Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like gamma, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after alpha- and gamma-irradiation were comparable, though the dose-response relationships differed. The "RBE" value for G2M block for alpha- versus gamma-radiation was approx. 4. Electron microscopic studies of the cell samples where a large proportion of cells were arrested in G2M showed subcellular changes in nuclear membrane and the presence of morphologically apoptotic cells. Biochemical analysis of DNA from irradiated cells by agarose gel electrophoresis revealed more extensive DNA fragmentation for alpha- vs gamma-irradiation, even at relatively low total doses. We conclude that the high LET radiation is more efficient in inducing G2M block and apoptosis in EL4 lymphoma cells. The overall radiosensitivity of some high and low grade malignant lymphoma cells to radiation may correlate with these processes. The clinical implications of 212Bi-induced G2M delay may be particularly important for biologically targeted high LET radiopharmaceutical therapy.

Preparation and evaluation of para-[211At]astatobenzoyl labeled anti-renal cell carcinoma antibody A6H F(ab')2. In vivo distribution comparison with para-[125I]iodobenzoyl labeled A6H F(ab')2.

A preliminary investigation of an 211At labeled anti-renal cell carcinoma antibody fragment, A6H F(ab')2, was conducted. In the investigation, A6H F(ab')2 was labeled by conjugation with N-succinimidyl p-[211At]astatobenzoate, and the in vivo biodistribution was evaluated in athymic mice bearing TK-82 renal cell carcinoma xenografts. As a control, p-[125I]iodobenzoyl labeled A6H F(ab')2 was coinjected with the astatinated F(ab')2. The data obtained demonstrated that the two radiolabels (211At and 125I) had quite similar distributions, providing evidence that the 211At remained attached to the A6H F(ab')2 in vivo. Further, the astatinated antibody attained a 2:1 tumor-to-blood ratio, and greater than 35:1 tumor-to-muscle ratio, at 4h post-injection, suggesting that this antibody conjugate could be used to evaluate treatment of metastatic renal cell carcinoma in a mouse model.

The effects of radon daughter alpha-particle irradiation in K1 and xrs-5 CHO cell lines.

We investigated the radiobiological effects of the radon daughter bismuth-212 (212Bi) in Chinese hamster ovary (CHO) K1 cells and in xrs-5 cells, which are X-ray sensitive and deficient in the ability to rejoin DNA double-strand breaks. The cells were exposed to 250 kVp X-rays or to 212Bi chelated to diethylene triamine pentaacetic acid (DTPA); chelation of 212Bi to DTPA prevented its attachment to or entry into the cells. Cytotoxic, clastogenic, and mutagenic responses of the cells were measured and RBEs (D10, 2 chromatid aberrations/cell and 10 induced 6-thioguanine-resistant mutants) were calculated to be 3.8, 3.5, and 3.9, respectively for K1, and 1.4, 0.8, and 5.1, respectively, for xrs-5. With the exception of the RBE of less than 1 for alpha-induced aberrations in xrs-5, the results are consistent with the following conclusions: (1) alpha-particles are in general more effective cytotoxic, clastogenic and mutagenic agents than X-rays; (2) the primary lethal and clastogenic lesion induced by both X-rays and alpha-particles is probably a DNA double-strand break; (3) DNA double-strand breaks induced by alpha-radiation are less well repaired than those induced by X-rays, although a portion of alpha-induced damage is repairable; and (4) deficiencies in rejoining DNA double-strand breaks affect the clastogenic and cytotoxic effects of X-rays and alpha-radiation, not their mutagenic effects. The RBE of 0.8 for aberration induction in xrs-5 cells could reflect a deficiency in the ability of these cells to convert alpha-induced damage to chromosome aberrations. Alternatively, the RBE of less than 1 might reflect an unusual sensitivity of xrs-5 cells to alpha-induced G2 delays.

Interlaboratory comparison of different alpha-particle and radon sources: cell survival and relative biological effectiveness.

Alpha radiation-induced cell killing was determined in four different laboratories in order to: 1) measure interlaboratory variability and 2) compare the effects of radon and radon daughter exposures with the effects of 238Pu (an often-used model for radon exposure). The results suggest that differences in handling from laboratory to laboratory can affect both low and high linear energy transfer responses and should be considered when comparing results from different laboratories.