High-linear energy transfer (LET) alpha versus low-LET beta emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and dose-limiting toxicity of 213Bi- versus 90Y-labeled CO17-1A Fab' fragments in a human colonic cancer model.

Recent studies suggest that radioimmunotherapy (RIT) with high-linear energy transfer (LET) radiation may have therapeutic advantages over conventional low-LET (e.g., beta-) emissions. Furthermore, fragments may be more effective in controlling tumor growth than complete IgG. However, to the best of our knowledge, no investigators have attempted a direct comparison of the therapeutic efficacy and toxicity of a systemic targeted therapeutic strategy, using high-LET alpha versus low-LET beta emitters in vivo. The aim of this study was, therefore, to assess the toxicity and antitumor efficacy of RIT with the alpha emitter 213Bi/213Po, as compared to the beta emitter 90Y, linked to a monovalent Fab' fragment in a human colonic cancer xenograft model in nude mice. Biodistribution studies of 213Bi- or 88Y-labeled benzyl-diethylene-triamine-pentaacetate-conjugated Fab' fragments of the murine monoclonal antibody CO17-1A were performed in nude mice bearing s.c. human colon cancer xenografts. 213Bi was readily obtained from an "in-house" 225Ac/213Bi generator. It decays by beta- and 440-keV gamma emission, with a t(1/2) of 45.6 min, as compared to the ultra-short-lived alpha emitter, 213Po (t(1/2) = 4.2 micros). For therapy, the mice were injected either with 213Bi- or 90Y-labeled CO17-1A Fab', whereas control groups were left untreated or were given a radiolabeled irrelevant control antibody. The maximum tolerated dose (MTD) of each agent was determined. The mice were treated with or without inhibition of the renal accretion of antibody fragments by D-lysine (T. M. Behr et al., Cancer Res., 55: 3825-3834, 1995), bone marrow transplantation, or combinations thereof. Myelotoxicity and potential second-organ toxicities, as well as tumor growth, were monitored at weekly intervals. Additionally, the therapeutic efficacy of both 213Bi- and 90Y-labeled CO17-1A Fab' was compared in a GW-39 model metastatic to the liver of nude mice. In accordance with kidney uptake values of as high as > or = 80% of the injected dose per gram, the kidney was the first dose-limiting organ using both 90Y- and 213Bi-labeled Fab' fragments. Application of D-lysine decreased the renal dose by >3-fold. Accordingly, myelotoxicity became dose limiting with both conjugates. By using lysine protection, the MTD of 90Y-Fab' was 250 microCi and the MTD of 213Bi-Fab' was 700 microCi, corresponding to blood doses of 5-8 Gy. Additional bone marrow transplantation allowed for an increase of the MTD of 90Y-Fab' to 400 microCi and for 213Bi-Fab' to 1100 microCi, respectively. At these very dose levels, no biochemical or histological evidence of renal damage was observed (kidney doses of <35 Gy). At equitoxic dosing, 213Bi-labeled Fab' fragments were significantly more effective than the respective 90Y-labeled conjugates. In the metastatic model, all untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation, whereas a histologically confirmed cure was observed in 95% of those animals treated with 700 microCi of 213Bi-Fab' 10 days after model induction, which is in contrast to an only 20% cure rate in mice treated with 250 microCi of 90Y-Fab'. These data show that RIT with alpha emitters may be therapeutically more effective than conventional beta emitters. Surprisingly, maximum tolerated blood doses were, at 5-8 Gy, very similar between high-LET alpha and low-LET beta emitters. Due to its short physical half-life, 213Bi appears to be especially suitable for use in conjunction with fast-clearing fragments.     

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.     

Biodistribution and radiation dose estimates for yttrium- and iodine-labeled monoclonal antibody IgG and fragments in nude mice bearing human colonic tumor xenografts

An anti-carcinoembryonic antigen murine monoclonal antibody designated NP-4, and its F(ab')2 and Fab' fragments, were coupled to the 1/1 mixture of 1-isothiocyanato-benzyl-3-methyl- and 1-methyl-3-isothiocyanato-benzyl-diethylenetriaminepentaacetic acid chelate and labeled with 111In or 88Y. Biodistribution studies in nude mice bearing a human colonic tumor xenograft were performed with these labeled conjugates, and comparisons were made to unconjugated NP-4 IgG and fragments labeled with 131I. Regardless of the labeling method, higher tumor uptake was found with the intact IgG than with the fragments, but due to faster blood clearance, tumor/blood ratios were higher for the fragments than for the IgG. Tumor uptake for the radiometal-labeled NP-4 was generally higher than the 131I-labeled NP-4. Tumor/nontumor ratios for the liver, kidney, and spleen were higher for the 111In- and 88Y-labeled NP-4 IgG than the respective radiometal-labeled fragments, but tumor/nontumor ratios for the 131I-NP-4 fragments were higher than the 131I-NP-4 IgG. Radiometal uptake in the kidney was approximately 8 and 150 times higher than the 131I-NP-4 F(ab')2 and Fab', respectively, and the clearance of radiometal activity in the kidneys was approximately 10 times slower than the radioiodine. Quantitation of 88Y or 111In activity in the femur showed 3-5%/g for the IgG and F(ab')2 and only 1-2%/g for the Fab'. The amount of radioactivity in the femur remained constant over time, and between 60 and 100% of the 88Y activity remained after flushing the core of the femur with saline, whereas 50-70% of the 111In and only 25-30% of the 131I activity remained after washing. Radiation dose estimates derived from these studies suggest that at the maximal tolerated dose 131I-NP-4 IgG would deliver 5.9 times the dose to the tumor as 90Y-labeled NP-4 IgG. 90Y-labeled fragments would not be useful due to higher doses to the kidneys than to the tumor. However, with 131I-labeled IgG and fragments there is greater flexibility to permit tumoricidal doses without excessive toxicity to the normal tissues.     

Radioimmunotherapy of human colorectal carcinoma xenografts using 90Y-labeled monoclonal antibody CO17-1A prepared by two bifunctional chelate techniques

Monoclonal antibody CO17-1A, which has specificity for colorectal and pancreatic carcinomas, was radiolabeled with the pure beta emitter, 90Y, by either the cyclic diethylenetriaminepentaacetic acid (DTPA) anhydride technique or by a site-specific bifunctional chelate technique using 1-(p-aminobenzyl)DTPA (p-NH2-Bz-DTPA). Female nude mice bearing SW 948 human colorectal carcinoma xenografts were given injections i.v. of 90Y-labeled monoclonal antibody CO17-1A at dosages of 100, 150, and 200 muCi/25 g body weight. Unlabeled CO17-1A (100 micrograms/25 g body weight) was coadministered. In animals receiving 90Y-CO17-1A prepared by the cyclic DTPA anhydride technique, tumor volume was unchanged from base line at a dose of 200 microCi/25 g. As the dosage of 90Y-CO17-1A increased, the rate of tumor growth decreased, but all experimental animals in this group died between 14 and 21 days. In contrast, CO17-1A radiolabeled with 90Y by the site-specific p-NH2-Bz-DTPA bifunctional chelate technique produced a maximum tumor volume reduction of 87% in the 200 microCi/25 g group by day 15, and no deaths were noted in any of the 90Y-CO17-1A-treated groups for 71 days. Dose-response curves again showed increased tumoricidal effects with increased dosages of 90Y-CO17-1A. S-2-(3-Aminopropylamino)ethylphosphorothioic acid, commonly known as WR-2721, is a radioprotective drug which has been shown to protect against bone marrow depression in irradiated humans. No protection was observed when WR-2721 was used as an adjunct to treatment with 90Y-CO17-1A prepared by either the cyclic DTPA anhydride technique or the site-specific p-NH2-Bz-DTPA technique. When the site-specific p-NH2-Bz-DTPA technique was used, the reduction in WBC and hemoglobin levels correlated with increasing bone marrow toxicity at higher doses. We conclude that CO17-1A labeled with 90Y via the site-specific p-NH2-Bz-DTPA technique has potential for radioimmunotherapy of human colorectal carcinoma.     

A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles

PURPOSE: Integrin alpha(v)beta(3) and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated alpha(v)beta(3) and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models. METHODS AND MATERIALS: The treatment regimens used nanoparticle (NP) based targeting agents radiolabeled with (90)Y. The small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoethylsulfonylamino-beta-alanine, which binds to the integrin alpha(v)beta(3), and a monoclonal antibody against murine Flk-1 (anti-Flk-1 MAb) were used to target the NPs. Murine tumor models K1735-M2 (melanoma) and CT-26 (colon adenocarcinoma) were used to evaluate the treatment efficacy. RESULTS: In K1735-M2 and CT-26 tumors, a single treatment with IA-NP-(90)Y (14.2 microg/g IA, 5 or 6 microCi/g (90)Y) caused a significant tumor growth delay compared to untreated control tumors, as well as tumors treated with IA, IA-NP, and NP-(90)Y, respectively (p < 0.025, Wilcoxon test). In K1735-M2 tumors, a single treatment with anti-Flk-1 MAb-NP-(90)Y (0.36 microg/g anti-Flk-1 MAb, 5 microCi/g (90)Y) also caused a significant tumor growth delay (p < 0.05, Wilcoxon test) compared to untreated tumors, as well as tumors treated with anti-Flk-1 MAb, anti-Flk-1 MAb-NP, and conventional radioimmunotherapy with (90)Y-labeled anti-Flk mAb. Anti-CD31 staining showed a marked decrease in vessel density in tumors treated with anti-Flk-1 MAb-NP-(90)Y, which was associated with a high level of apoptotic death in these tumors, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. CONCLUSIONS: The present studies provide proof of principle that targeted radiotherapy works using different targeting agents on a nanoparticle, to target both the integrin alpha(v)beta(3) and the vascular endothelial growth factor receptor. These encouraging results demonstrate the potential therapeutic efficacy of the IA-NP-(90)Y and anti-Flk-1 MAb-NP-(90)Y complexes as novel therapeutic agents for the treatment of a variety of tumor types.     

Preclinical studies targeting normal and leukemic hematopoietic cells with Yttrium-90-labeled anti-CD45 antibody in vitro and in vivo in nude mice

A study was undertaken to investigate the suitability of using a high affinity (Kd = 1.1 nM) anti-CD45 monoclonal antibody for delivering the high energy beta-particle emitting isotope (90)Y to lymphohematopoietic target cells in vivo. The antibody, AHN-12, recognized the tyrosine phosphatase CD45 expressed on the surface of normal and malignant hematopoietic cells and studies showed that it reacted with both CD45-expressing normal peripheral blood cells and leukemia cells from patients. The antibody was readily labeled with (90)Y using the highly stable chelate 1B4M-DTPA and the radioimmunoconjugate was designated (90)Y-anti-CD45. The agent selectively bound to CD45(+) B cell line Daudi, but not CD45(-) control cells and significantly (p = 0.007) more bound to Daudi tumors growing in athymic nude mice than did a control non-reactive antibody. Moreover, biodistribution data correlated well to an anti-Daudi effect observed against established tumors in nude mice. The effect was dose dependent and irreversible with the best results in mice receiving a single dose of 137 microCi (90)Y-anti-CD45. These mice displayed a significantly (p < 0.0095) better anti-tumor effect than a control (90)Y-labeled antibody and survived over 135 days with no evidence of tumor. Histology studies showed no significant injury to kidney, liver, or small intestine even at 254 microCi, the highest dose tested. Because radiolabeled anti-CD45 antibody can be used to deliver radiation selectively to lymphohematopoietic tissue, these data indicate that this agent may be used to improve treatment of hematopoietic malignancies, particularly leukemia and lymphoma, when combined with hematopoietic stem cell transplantation in a future clinical trial.     

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.     

Specific radioimmunotherapy using 90Y-labeled monoclonal antibody in erythroleukemic mice

Radioimmunotherapy using 90Y-labeled diethylenetriamine pentaacetic acid-antibody conjugates was studied in Rauscher erythroleukemia virus-infected mice. Preliminary experiments showed that biodistribution profiles for nonrelevant mouse monoclonal antibody and polyclonal bovine immunoglobulin were identical in both normal and leukemic mice. Therefore, bovine immunoglobulin G was selected as the control immunoglobulin in order to permit comparison to current clinical trials of radioimmunotherapy regimens. Specific monoclonal antibody was two- to three-fold more potent than bovine immunoglobulin G in therapy, as assessed by reduction of splenomegaly (dose required for half-maximal effect, 9 microCi versus 16 to 27 microCi). Mice treated with 50 microCi 90Y-labeled control immunoglobulin had spleens which were twice the normal size and showed extensive areas of erythropoiesis indicative of the presence of tumor foci; in contrast, doses as low as 27 microCi 90Y-labeled specific antibody resulted in complete remission with no microscopic evidence of tumor foci in either spleen or liver. Although reversible marrow toxicity was observed it was not dose limiting. These results demonstrate that tumor-specific therapy is possible using 90Y-labeled antibody.     

Preclinical assessments of 90Y-labeled C110 anti-carcinoembryonic antigen immunotoxin: a therapeutic immunoconjugate for human colon cancer

We have synthesized 90Y-labeled immunotoxin (IT) containing ricin A chain and C110 anti-carcinoembryonic antigen monoclonal antibody (MAb) to produce a therapeutic immunoconjugate for human colon cancer. The C110 IT was labeled with 90Y via a benzylisothiocyanate derivative of diethylenetriaminepentaacetic acid. The efficiency of 90Y labeling was consistently 90 to 98%, with a specific activity of about 1 microCi/microgram. In in vitro stability studies, more than 80% of 90Y remained bound to the C110 IT for up to 5 days after incubation. The percentage of binding of 90Y-labeled C110 IT to carcinoembryonic antigen-coated microbeads was 86%, indicating good retention of the initial immunoreactivity of the C110 MAb. In in vitro protein synthesis inhibition assays, 90Y-labeled C110 IT was approximately 3.7-fold more toxic to the LS174T human colon carcinoma cell line than unmodified C110 IT and 1380-fold more toxic than 90Y-labeled C110 MAb. Biodistribution studies of 90Y-labeled C110 IT in LS174T tumor-bearing mice showed that, at 24 h following i.p. injection, high accumulation of radioactivity was seen in the i.p. tumor and liver and, thereafter, high accumulation in these tissues remained almost unchanged until up to 168 h, with percentage of injected dose/g ranging from 15 to 18% in the tumor and 10 to 15% in the liver. The radioactivity in the spleen and bone gradually increased with time and reached their highest levels (approximately 8% of injected dose/g) at 168 h. Estimation of absorbed radiation doses to the tissues showed that i.p. tumor would have received an approximately 1.5 to 7 times higher radiation dose than normal organs. In in vivo therapeutic trials, 90Y-labeled C110 IT provided survival prolongation of LS174T tumor-bearing mice superior to that with either unmodified C110 IT or 90Y-labeled C110 MAb (4 less than 0.01; Mann-Whitney U test). These results indicate that 90Y-labeled C110 anti-carcinoembryonic antigen IT may be a potent therapeutic immunoconjugate for human colon cancer and that it may have direct relevance for i.p. treatment of peritoneal carcinomatosis from colon cancers.     

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery.

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non-specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross-linking of reduced Fab' fragments from PR1A3 and T84.66. Fifty-nine tumors from 2 human colorectal carcinoma cell lines with high (KM-12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I-labeled MAbs: IgG, F(ab')(2) and Fab' of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG-pretreated mice and the least in biparatopic MAb-pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG-pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3-pretreated mice and 2 to 4.6 in biparatopic MAb-pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb-pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb-pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG-pretreated mice (50% and 72%). The biparatopic MAb using 2 anti-CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.     

Effect of yttrium-90-labeled anti-carcinoembryonic antigen monoclonal antibody on the morphology and phenotype of human tumors grown as peritoneal carcinomatosis in athymic mice

Grossly visible peritoneal carcinomatosis resembling that seen in man was produced in athymic mice 7 days after intraperitoneal injection of 8 x 10(5) cells of the carcinoembryonic antigen (CEA)-producing human colon carcinoma cell line LS174T. The mice received intraperitoneal injections of 40 to 160 microCi of yttrium-90 (90Y)-labeled anti-CEA monoclonal antibody (MAb). When the mice were killed 12 days after injection, a significant inhibition of tumor growth, ranging from 40% to 95%, was observed in the treated animals when compared to the growth of tumors in the untreated animals (P less than 0.001). No mortality secondary to the therapy was seen. The bone marrow was depleted significantly at the higher doses of labeled MAb, but total recovery was observed 4 weeks after treatment. Histologically, the treated tumors showed extensive radiation effects early in the posttherapy period and massive necrosis at later times. Minute foci of viable tumor remained in the periphery. New tumor outgrowths with histologic features similar to those in the untreated controls began to appear 3 weeks after therapy. The CEA expression of the treated tumors was similar to that of the untreated controls during the early posttreatment period, diminishing progressively as the tumors became necrotic. Newly grown tumor nodules in the treated animals lacked significant CEA expression both initially and at later times. Our studies suggest that therapy with 90Y-anti-CEA MAb therapy results in selection of tumor clones lacking CEA, and that a single large dose of 90Y-MAb should be more effective than multiple fractions of smaller doses.     

Yttrium-90 and iodine-131 radioimmunoglobulin therapy of an experimental human hepatoma

Therapeutic trials were performed on the HepG2 human hepatoblastoma implanted s.c. in the athymic nude mouse. Animals were treated with polyclonal and monoclonal antiferritin and control antibodies labeled with either iodine-131 (131I) or yttrium-90 (90Y). Administration of 400 muCi of 131I-labeled polyclonal antiferritin or 300 muCi of 90Y-labeled polyclonal antiferritin significantly increased survival (P less than 0.001). There were no tumor cures with radiolabeled polyclonal antibody therapy. Animals treated with 200 or 300 muCi of 131I-labeled monoclonal antiferritin (QCI054) did not show increased survival compared to controls. Although 400 muCi of 131I-labeled QCI significantly prolonged survival, treatment resulted in no long-term survivors. Monoclonal antiferritin labeled with 90Y significantly prolonged survival of animals (P less than 0.001) at doses of 100, 200, or 300 muCi compared with untreated controls. Fifty % of the animals treated with 200 muCi and 75% of the animals treated with 300 muCi showed no evidence of disease at 140 days following treatment. Four hundred muCi of 90Y-labeled QCI proved toxic to the animals. Increased survival was accompanied by a decrease in tumor mitotic rate and increase in cellular polymorphism as determined by pathological examination. The radiation dose absorbed in the tumor correlated directly with tumor response following treatment. The absorbed dose in tumors for complete decay of the isotope ranged from 165 and 330 cGy at the periphery and center of small tumors for an administered activity of 200 muCi of 131I-labeled polyclonal antiferritin, to 7,573 and 12,400 cGy for 300 muCi of 90Y-labeled monoclonal antiferritin QCI.     

Applicability of carcinoembryonic antigen-specific monoclonal antibodies to radioimmunoguided surgery for human colorectal carcinoma

Two carcinoembryonic antigen (CEA)-specific monoclonal antibodies (MAbs), PR1A3 and T84.66, were tested to determine whether they could accurately localize colorectal carcinoma and therefore be applicable in radioimmunoguided surgery (RIGS). Twenty-one tumors by three human colorectal carcinoma cell lines with various levels of CEA expression (KM-12c, C75, and Clone A) were successfully implanted in the intra-abdominal organs of 15 nude mice. The tumors was localized using a portable radioisotope detector (Neoprobe 1000) 48 h after injection of radiolabeled MAbs (10 mCi/mouse) when the precordial counts were <20 per 2 s. Histopathological identification of radiolabeled MAbs were also performed using immunohistochemistry and microautoradiography. Radioactivity counted on a portable radioisotope detector correlated well with that on a gamma counter. The distribution in the blood was significantly greater than in other organs (P < 0.001). Localization indices of the tumor in various organs was from 1.1 to 8.5 in the PR1A3-pretreated mice and 3.0 to 8.6 in the T84.66-pretreated mice. Silver grains and immune staining were distributed in the tumor cells of the PR1A3-pretreated mice, whereas they were in the necrotic debris as well as the tumor cells of the T84.66-pretreated mice. There were significantly more silver grains in the liver in the T84.66-pretreated mice than in the PR1A3-pretreated mice (P = 0.004). The sensitivity and specificity of tumor localization by RIGS were 71.4 and 91.4% in the PR1A3-pretreated mice, whereas they were 60 and 76% in the T84.66-pretreated mice. A study using specific anti-CEA MAbs suggested PR1A3 as an efficient immune probe for RIGS in colorectal carcinoma with a low rate of false-positive detection.     

Selection of a DTPA chelate conjugate for monoclonal antibody targeting to a human colonic tumor in nude mice

Our previous studies with a 90Y-labelled antibody against carcinoembryonic antigen (CEA) conjugated to the cyclic anhydride-DTPA (CA-DTPA) indicated that the accretion of 90Y in the bone may limit the application of 90Y-labelled antibodies for therapy. In this report, we have compared the tumor targeting of CA-DTPA-conjugated antibody to antibody conjugated with 4 isothiocyanatobenzyl (ITC-Bz) derivatives of DTPA in nude mice bearing a human colonic tumor xenograft. In biodistribution studies using an 111In-labelled anti-CEA murine monoclonal antibody (MAb), the CA-DTPA-conjugated MAb showed lower tumor uptake, faster blood clearance, and higher accretion in the liver than any of the 4 ITC-Bz-DTPA-conjugated MAbs. There were smaller differences among the 4 ITC-Bz-DTPA conjugates. Whole-body autoradiography of animals given 90Y-MAb prepared with the CA-DTPA or the ITC-Bz-DTPA showed less radioactivity in the bone with the ITC-Bz-DTPA-MAb than the CA-DTPA-MAb. 90Y uptake in the bone corresponded with regions of low proliferative activity as defined by 3H-labelled thymidine, suggesting that the 90Y was in the cortex rather than the marrow. These studies clearly show an advantage of the ITC-Bz-DTPA derivatives for 90Y and 111In labelling of MAbs.     

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of the T cell lymphoma EL4

Yttrium-90 is a potent beta-emitting radionuclide with potential for therapy of lymphoma. A monoclonal antibody against Ly1, the murine homologue of human CD5, was labeled with 90Y and found to selectively bind to Ly1-positive, radiation-sensitive, EL4 mouse lymphoma cells. When tested in this aggressive model of T cell lymphoma, in vivo studies in C57BL/6 mice showed that a single 140-microCi i.p. dose of 90Y-anti-Ly1, given 1 day after i.v. injection of a lethal dose of EL4 cells, resulted in significant but transient improvement in survival. Protection was selective, since a 90Y-labeled irrelevant control antibody did not prolong survival. Biodistribution studies showed that protection was likely limited by inadequate localization of labeled antibody to tumor. Importantly, labeled anti-Ly1 specifically localized in the immunological tissue (spleen and thymus) and lowered the WBC count, perhaps limiting the tolerated dose. Myelosuppression, which is considered one of the major side effects associated with 90Y usage, was not a lethal complication, since WBC counts recovered in mice given a 140-microCi dose of 90Y-anti-Ly1 without EL4 cells and 100% of these animals survived. The maximum tolerated dose was less than 200 microCi. Despite the high localization of 90Y-anti-Ly1 in spleen, splenectomies of tumor-injected mice did not improve the antitumor efficacy of radiolabeled antibody. Further evidence for inadequate delivery of radionuclide to tumor was shown when external total-body irradiation was given to mice given injections of a lethal dose of EL4 tumor cells. Comparison of internal and external irradiation studies indicated that the partially protective effect of 140 microCi 90Y-Ly1 was equivalent to external radiation of only 100-200 cGy. Because this model reflects the current clinical limitations of radiolabeled antibodies for therapy, including partial antitumor efficacy, delivery of labeled anti-T cell antibodies to the immune system, and low maximum tolerated dose, the model may be useful for examining strategies which could increase the tolerated dose and therapeutic efficacy.     

Carcinoembryonic antigen expression of resurgent human colon carcinoma after treatment with therapeutic doses of 90Y-alpha-carcinoembryonic antigen monoclonal antibody

We have previously shown that the colon carcinoma (LS174T) xenografts that emerged shortly after radioimmunotherapy with 90Y-labeled anti-CEA monoclonal antibody (MAb) ZCE025 lacked significant expression of CEA in comparison with the untreated tumors. The present study was designed to establish if the immunophenotype of the treated tumors was the result of CEA specific therapy and if the effect was permanent. Athymic mice bearing LS174T tumors were treated either with 120 mu Ci of 90Y-ZCE025, an equal dose of 90Y-96.5 (nonspecific MAb), or received no treatment. When the treated tumors grew to approximately 1.5 cm in diameter (6 weeks after therapy), they were resected and aliquoted to be transplanted to other mice, plated in tissue culture, fixed in formalin, and homogenized for CEA quantitation. The procedure was repeated 3 times (a total of 4 months after treatment). The CEA content was evaluated 2 and 6 weeks after therapy and when the tumors were transplanted. We confirmed a 4-fold decrease of CEA in the resurgent tumors 6 weeks after specific 90Y-ZCE025 therapy, which was twice the decrease experienced by the tumors treated with nonspecific 90Y-96.5, indicating substantial and specific killing of CEA-expressing cells. The CEA content slowly but progressively increased with each new pass of the tumor in the mice, reaching approximately one-half the value of the controls at the end of the study. The resurgent tumors were also studied by immunohistochemistry with MAbs detecting different epitopes of CEA, keratin, TAG-72, and epithelial membrane antigen to evaluate possible additional immunophenotypic changes induced by radioimmunotherapy. Only the expression of TAG-72 (recognized by MAb B72.3) increased immediately after therapy, but it returned to the original levels by the end of the study. These results suggest that: (a) specific radioimmunotherapy with 90Y-ZCE025 selectively kills cells that express higher levels of CEA; (b) the immunophenotype of the surviving fraction of the tumor appears to slowly revert to its original form; and (c) other tumor markers unrelated to CEA can also be affected. These observations have important implications for the design of radioimmunotherapy trials.     

Radioimmunotherapy of CD22-expressing Daudi tumors in nude mice with a 90Y-labeled anti-CD22 monoclonal antibody.

A study was undertaken to investigate the efficacy of a high affinity, rapidly internalizing anti-CD22 monoclonal antibody for selectively delivering high-energy (90)Y radioactivity to B lymphoma cells in vivo. The antibody, RFB4, was readily labeled with (90)Y using the highly stable chelate, 1B4M-diethylenetriaminepentaacetic acid. Labeled RFB4 selectively bound to the CD22(+) Burkitt's lymphoma cell line Daudi, but not to CD22(-) control cells in vitro as compared with a control antibody, and was more significantly bound (P = 0.03) to Daudi solid tumors growing in athymic nude mice. Biodistribution data correlated well with the antitumor effect. The therapeutic effect of (90)Y-labeled anti-CD22 (Y22) was dose-dependent, irreversible, and the best results were achieved in mice receiving a single i.p. dose of 196 microCi. These mice displayed a significantly better (P < 0.01) antitumor response than control mice and survived >200 days with no evidence of tumor. Histology studies showed no significant injury to kidney, liver, or small intestine. Importantly, tumor-bearing mice treated with Y22 had no radiologic bone marrow damage compared with tumor-bearing mice treated with the control-labeled antibody arguing that the presence of CD22(+) tumor protected mice from bone marrow damage. When anti-CD22 radioimmunotherapy was compared to radioimmunotherapy with anti-CD19 and anti-CD45 antibodies, all three antibodies distributed significantly high levels of radioisotope to flank tumors in vivo compared with controls (P < 0.05), induced complete remission, and produced long-term, tumor-free survivors. These findings indicate that anti-CD22 radioimmunotherapy with Y22 is highly effective in vivo against CD22-expressing malignancies and may be a useful therapy for drug-refractory B cell leukemia patients.     

Radioimmunotherapy of A431 xenografted mice with pretargeted B3 antibody-streptavidin and (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin

We investigated the biodistribution of (88)Y/(111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin and therapy with (90)Y-labeled DOTA-biotin in tumor-bearing mice after B3-streptavidin antibody conjugate (B3-SA) pretargeting. B3 antibody, recognizing Lewis(y) antigen, was conjugated to streptavidin (B3-SA). For pretargeting, 400 micro g of the B3-SA was injected i.v. into mice bearing A431 tumor xenografts. After tumor localization of B3-SA, 100 micro g of synthetic clearing agent was injected i.v. to clear the unbound B3-SA from the circulation. Four h later, 1 micro g of radiolabeled DOTA-biotin was injected i.v. Radioimmunotherapy was performed with doses of 9.25 to 37 MBq of (90)Y-labeled DOTA-biotin. As a result, radiolabeled DOTA-biotin cleared rapidly. All of the normal tissues had <2.6% of the injected dose per gram, whereas tumor uptake reached approximately 15% ID/g. The total tumor uptake of radioactivity remained similar for 96 h or longer. In the first study, the median survival of the control group was 8 days, whereas it increased to >163 days in the 37 MBq (90)Y group (P < 0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of (90)Y and B3-SA were cured, and remained healthy for >180 days after therapy, compared with control groups, with 相似文献   

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.     

Human carcinoembryonic antigen cDNA expressed in rat carcinoma cells can function as target antigen for tumor localization of antibodies in nude rats and as rejection antigen in syngeneic rats.

We have tried to develop a new model consisting of rats transplanted with syngeneic colon carcinoma PROb cells transfected with cDNA coding for the carcinoembryonic antigen (CEA), the human tumor marker most commonly used as target for MAbs. The antigenic density of the 4 CEA-expressing clones selected for a precise characterization ranged from 5 x 10(4) to 1 x 10(6) CEA molecules per cell. In all clones the CEA was shown to be attached to the membrane by a phosphatidylinositol (PI) anchor. Using a panel of radiolabeled MAbs directed against the 5 major epitopes described on the CEA molecule, we showed that all these CEA epitopes were expressed by the 4 transfectants. Southern-blot analysis showed that the entire CEA cDNA was present in the transfectants. Western-blot analysis, however, showed that the size of the CEA expressed by the 4 transfectants was slightly smaller than that of CEA produced by 2 reference human colon-carcinoma cell lines. Two clones, expressing 1 x 10(5) and 1 x 10(6) CEA molecules per cell, respectively, were grafted s.c. in nude mice and rats. Injection of radiolabeled anti-CEA F(ab')2 fragments into these animals showed specific tumor localization with the highest percentages of injected doses for the transfectants expressing the highest CEA level. When grafted into immunocompetent syngeneic BDIX rats, the CEA-expressing clones induced a strong antibody response against CEA and tumor rejections in a majority of the animals. Although the analysis of the immune response against the CEA-cDNA-transfected carcinoma cells is under investigation, the present results demonstrate that human CEA could function as a rejection antigen when transfected into rat carcinoma cells.