Enhanced delivery of a monoclonal antibody F(ab')2 fragment to subcutaneous human glioma xenografts using local hyperthermia

The purpose of this study was to investigate the effects of tumor-localized hyperthermia at 42 degrees C on the tissue distribution of radioiodinated monoclonal antibody F(ab')2 fragments. Paired-label biodistribution measurements were performed in athymic mice bearing D-54 MG human glioma xenografts on one leg. Mice received both the 131I-labeled F(ab')2 fragment of Mel-14, reactive with human gliomas and melanomas, and nonspecific 125I-labeled RPC 5 F(ab')2. Tumor-bearing legs were placed in a 42 degrees C water bath or a 37 degrees C water bath (control) for 2 or 4 h. In mice sacrificed immediately after 2 h of heating, no hyperthermia-induced differences in the distribution of either fragment were observed. In the 4-h groups, tumor uptake of Mel-14 F(ab')2 increased from 7.04 +/- 1.59% injected dose (ID)/g at 37 degrees C to 20.65 +/- 4.53% ID/g at 42 degrees C (P less than 0.0001), and tumor localization of the control fragment rose from 5.23 +/- 1.35% ID/g to 14.51 +/- 1.37% ID/g (P less than 0.0001). In another experiment, F(ab')2 fragments were injected, tumors were heated for 4 h, and groups were sacrificed at 4, 8, and 16 h after injection. Statistically significant 2- to 3-fold higher uptake of both fragments in tumor were observed at all time points. Hyperthermic conditions also resulted in higher tumor:tissue ratios for both fragments. These results suggest that it may be possible to use tumor-localized hyperthermia to increase the therapeutic utility of radiolabeled monoclonal antibodies, particularly when labeled with short lived nuclides such as the 7.2-h alpha-emitter 211At.     

Comparative localization of murine monoclonal antibody Me1-14 F(ab')2 fragment and whole IgG2a in human glioma xenografts

Monoclonal antibodies (MAbs) targeted to glioma-associated antigens may allow the selective delivery of imaging and therapeutic agents to brain tumors; the use of MAb fragments may be a strategy to further improve tumor uptake of such agents relative to normal tissues. In this study, we have examined the in vivo localization of radioiodinated MAb Me1-14, a murine immunoglobulin G2a (IgG2a) reactive with gliomas, and its F(ab')2 fragment in s.c. and intracranial xenografts of human glioma cell line D-54 MG in athymic mice. The radiolabeled F(ab')2 fragment of Me1-14 was demonstrated to possess in vitro binding affinity and immunoreactivity comparable to that of whole IgG. Direct comparison of IgG and F(ab')2 biodistribution in s.c. xenograft-bearing mice showed higher tumor: normal tissue ratios of the F(ab')2 fragment compared to IgG. In intracranial tumor-bearing mice paired-label analysis using a nonspecific protein control showed earlier specific tumor localization by the F(ab')2 fragment of Me1-14 compared to IgG. Blood-to-tumor transfer constants (K1) derived for Me1-14 F(ab')2 were significantly greater than those for whole Me1-14 (P = 0.01). Estimated radiation dosimetry revealed that 131I-labeled Me1-14 F(ab')2 would deliver higher radiation doses to tumor than to normal tissues. These studies demonstrate that the F(ab')2 fragment of Me1-14 may be a potential agent for immune-directed brain tumor diagnosis and therapy.     

Monoclonal antibody and F(ab')2 fragment delivery to tumor in patients with glioma: comparison of intracarotid and intravenous administration

Non-i.v. delivery of radiolabeled monoclonal antibodies (MAbs) has been shown to increase tumor uptake and decrease dose to normal tissues. In this study, we have examined the potential advantage of intracarotid (i.c.) versus i.v. administration for the delivery of an intact MAb and a F(ab')2 fragment to tumor in patients with gliomas. Three patients received 10-50 mg of 81C6 IgG2b, a MAb reactive with the glioma-associated extracellular matrix antigen tenascin, and three received 5-20 mg of the F(ab')2 fragment of Mel-14, which is reactive with gliomas and melanomas. Paired-injection protocols, in which one-half of the MAb was labeled with 131I and administered by i.c. injection, and one-half was labeled with 125I and simultaneously administered by i.v. injection, were used. For both 81C6 IgG2b and Mel-14 (Fab')2, no differences in blood clearance half-times or urinary excretion rates of radioiodine were observed between i.c.- and i.v.-administered activity. Analysis of biopsy samples revealed i.c.:i.v. uptake ratios of 1.02 +/- 0.04, 0.95 +/- 0.03, and 1.03 +/- 0.05 for the accumulation of 81C6 IgG2b in temporalis muscle, normal brain, and glioma, respectively. Similarly, the i.c.:i.v. uptake ratios for Mel-14 F(ab')2 in these tissues were 0.98 +/- 0.04 (SD), 1.00 +/- 0.05, and 1.04 +/- 0.05. When the differences in percentage of injected dose/g uptake after i.c. and i.v. administration were compared, no statistically significant advantage for i.c. delivery was seen (P = 0.22-0.61). These data indicate that i.c. administration of MAb 81C6 IgG2b and Mel-14 F(ab')2 fragments offers no delivery advantage to offset the small but finite risk involved in cannulation and injection of the internal carotid artery.     

Experimental radioimmunotherapy of small peritoneal metastases of colorectal origin

Radioimmunotherapy using radiolabeled monoclonal antibodies (MoAbs) directed against tumor-associated antigens might be an effective treatment modality for small volume disease. Our aim was to optimize an experimental model of radioimmunotherapy for small peritoneal metastases of colorectal origin using the anti-CEA MoAb MN-14. In nude mice with intraperitoneal (i.p.) LS174T tumors, a protein dose-escalation study was carried out to determine the maximal dose of radioiodinated MN-14 to be used in radioimmunotherapy. The biodistribution of radioiodinated MN-14 was determined after intravenous (i.v.) and i.p. administration. Finally, the therapeutic efficacy of escalating activity doses of (131)I-labeled MN-14 (62.5-500 microCi) was assessed and compared to that of unlabeled MN-14 or 500 microCi of (131)I-labeled irrelevant control antibody. At protein doses higher than 25 microg, uptake in tumor was reduced, presumably due to saturation of tumor antigen. During the first 24 hours i.p. administration led to higher tumor uptake and higher tumor:blood ratios than i.v. administration. Median survival of the control groups was 38 days (unlabeled MN-14) and 52 days ((131)I-labeled nonspecific antibody). Median survival of the groups treated with increasing activity doses of (131)I-labeled MN-14 was 42 days (62.5 microCi), 49 days (125 microCi), 63 days (250 microCi) and 101 days (500 microCi), respectively (p < 0.0001 compared to unlabeled MN-14). The present study shows that the anti-CEA-antibody MN-14 preferentially accumulates in i.p. LS174T tumor xenografts after both i.p. and i.v. administration. Intraperitoneal radioimmunotherapy using (131)I-labeled MN-14 delays significantly the outgrowth of peritoneal LS174T metastases, even at relatively low activity doses.     

Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.

Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500-2000 microCi) or 131I-labeled CC49 IgG (250 and 500 microCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 microCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 microCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 microCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.     

Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells. In this study the F(ab')2 was the best compromise between the slowly cleared IgG and the poorly localized Fab in tumor imaging.     

A comparison of 131I-labeled monoclonal antibody 17-1A treatment to external beam irradiation on the growth of LS174T human colon carcinoma xenografts

Inhibition of growth of LS174T human colon cancer xenografts in athymic nude mice due to 131I-labeled MoAb 17-1A treatment was compared to inhibition due to different single doses of 60Co external radiation. From those data, conditions which produced equivalent radiobiological end points could be identified and compared to dose estimates calculated using a technique analogous to the Medical Internal Radiation Dose (MIRD) Committee formalism. The tumor growth rate in mice injected with a single intraperitoneal administration of 300 microCi of 131I-labeled MoAb was reduced relative to tumor growth in untreated control animals and in mice administered unlabeled MoAb and was found to be similar to the growth rate of tumors given a single 6 Gy dose of 60Co radiation. Furthermore, the growth rate of tumors in mice that received three injections of 300 microCi of 131I-labeled MoAb on days 9, 16 and 28 after tumor cell injection was similar to the growth rate of tumors given a single 60Co dose of 8 or 10 Gy. The biodistribution data for 125I-labeled 17-1A MoAb were used to calculate total doses for the tumor and various normal tissues in animals given a single administration of 131I-labeled 17-1A MoAb. The absorbed radiation dose in tumor was approximately five times higher than in normal tissues. The results of the present study indicate that the tumor growth inhibition produced by the administration of radiolabeled antibody can equal that produced by up to 10 Gy of external beam radiation. In addition, the MIRD calculations allow comparison of this form of low dose radiation to external photon irradiation.     

Localization of fluorine-18-labeled Mel-14 monoclonal antibody F(ab')2 fragment in a subcutaneous xenograft model.

Positron emission tomography is an imaging method that might improve the effectiveness of radioimmunoscintigraphy and might provide more accurate estimates of monoclonal antibody dosimetry prior to therapy. Because of its widespread availability, 2-h half-life 18F could be a useful nuclide for labeling monoclonal antibody fragments, provided that adequate tumor uptake and satisfactory tumor:normal tissue ratios could be achieved rapidly. In this study, the tissue distribution of 18F-labeled Mel-14 F(ab')2, a monoclonal antibody reactive with gliomas, was evaluated in a s.c. athymic mouse human glioma xenograft model. 18F labeling was performed using N-succinimidyl-8-(4-[18F]fluorobenzylamino) suberate. For paired-label comparisons both in vitro and in vivo, Mel-14 F(ab')2 was also labeled using N-succinimidyl 3- [125I]- iodobenzoate. When 100-120 micrograms of disuccinimidyl suberate was used in the 18F-labeled acylation agent synthesis, the binding of 18F-labeled Mel-14 F(ab')2 to glioma homogenates was comparable to that of the radioiodinated fragment. Scatchard analyses indicated nearly identical affinity constants for fragments with both labels (18F, 6.4 x 10(8) M-1; 125I, 6.7 x 10(8) M-1). Tumor levels of 18F increased between 1 and 2 h and then were relatively constant between 2 and 6 h. When lower levels of disuccinimidyl suberate were used, there was an excellent correlation between 18F and 125I tumor uptake (r = 0.984, slope 1.03-1.04). At 4 h, tumor:normal tissue ratios for 18F-labeled Mel-14 F(ab')2 in liver, spleen, muscle, and brain were 2.3, 4.2, 14, and 40, respectively. Localization indices, determined by comparison with 18F-labeled nonspecific F(ab')2, were 3.7 at 4 h and 6.9 at 6 h for tumor and about 1 for normal tissues, indicating the specificity of 18F-labeled Mel-14 F(ab')2 tumor uptake.     

Improved delivery of radiolabeled anti-B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody.

A human B-cell lymphoma xenograft model was used to test whether the administration of unlabeled MoAb prior to injection of radiolabeled monoclonal antibody (MoAb) improves delivery of the radiolabeled MoAb to tumor prior to testing in clinical radioimmunotherapy trials. The anti-B1/CD20 pan-B-cell MoAb reactive with human B-cell lymphomas and leukemias but not reactive with mouse B-cells was used in this study. Athymic nude mice bearing human Raji Burkitt lymphoma xenografts were given injections of 2.5 muCi (0.3 microgram) 131I-labeled anti-B1 with or without a 2-h prior single injection of 100 micrograms of unlabeled anti-B1 antibody. Four days later the animals given injections of 131I-labeled anti-B1 and the unlabeled anti-B1 predose had a tumor uptake of 12.72 +/- 1.17% (SEM) of injected dose/g which was 44% greater than the animals receiving the 131I-labeled anti-B1 alone (P = 0.014). The uptake in most normal tissues was unchanged, although the blood level of 131I-labeled anti-B1 appeared to be greater following unlabeled anti-B1 predosing (P = 0.067). Predosing with isotype matched irrelevant MoAb did not result in a greater tumor uptake or blood concentration of 131I-labeled anti-B1 compared to the administration of 131I-labeled anti-B1 alone. In studies using 111In-labeled anti-B1, the effect of unlabeled antibody predosing was more pronounced. For animals given injections of 4.5 muCi (0.4 microgram) 111In-labeled anti-B1 and the unlabeled anti-B1 predose, the uptake in tumor was 12.37 +/- 2.07% of injected dose/g which was 162% greater than the animals receiving the 111In-labeled anti-B1 alone (P = 0.009). Predosing decreased 111In-labeled anti-B1 uptake in spleen, while the blood level was significantly greater. Predosing was more effective than simultaneous injection in improving tumor delivery. When tumor-bearing mice were either simultaneously given injections of 36 micrograms of unlabeled anti-B1 and 4 micrograms 111In-labeled anti-B1 or were given preinjections of 36 micrograms unlabeled anti-B1 3 h prior to injection of 4 micrograms 111In-labeled anti-B1, tumor uptake 3 days later was 1.3-fold higher in the animals which received the preinjection of unlabeled antibody (P = 0.011). As the quantity of unlabeled anti-B1 was increased (36, 96, 996 micrograms) in the predose, significantly greater uptake in tumor was observed, although this uptake appeared to plateau at the highest predoses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiolocalization of human pancreatic tumors in athymic mice by monoclonal antibody DU-PAN 1

Monoclonal antibodies that selectively bind to pancreatic tumors may be useful in the therapy and diagnosis of pancreatic carcinoma. In this study we have examined the tumor localization of radioiodinated DU-PAN 1, a mouse monoclonal antibody that is selective for a human pancreatic cancer-associated antigen. After radiolabeling, both DU-PAN 1 intact monoclonal antibody and F(ab')2 fragments retained immunoreactivity and showed high affinity for the pancreatic tumor cell line CA13 in vitro. Paired-label biodistribution studies in nude mice bearing CA13 s.c. xenografts were performed. Mice received both 131I-labeled DU-PAN 1 immunoglobulin G2a or F(ab')2 fragment and 125I-labeled mouse myeloma immunoglobulin G2a or F(ab')2 fragment. Tumor uptake for 5-micrograms doses of DU-PAN 1 immunoglobulin ranged from 4.8 to 11.83% injected dose/g. Tumor uptake values for mice given 5-micrograms doses of DU-PAN 1 F(ab')2 ranged from 3.9 to 6.9% injected dose/g. Tumor uptakes of the respective myeloma controls were lower in all cases when compared with the DU-PAN 1 preparations. Tumor localization indices for 5-micrograms doses of DU-PAN 1 immunoglobulin were 3.0 and 24 h and 2.9 at 48 h. For 5-micrograms doses of DU-PAN 1 F(ab')2, tumor localization indices were 29.9 at 24 h and 90.0 at 48 h. In most cases, tumor:normal tissue ratios were greater than 3 at all time points, indicative of tumor selectivity for both DU-PAN 1 preparations, but the ratios were considerably higher using the DU-PAN 1 F(ab')2. The F(ab')2 fragment thus displays better tumor localization characteristics when compared with the intact immunoglobulin. Protein doses of DU-PAN 1 F(ab')2 of between 5 and 10 micrograms gave the best localization, although protein doses of up to 100 micrograms could be administered before apparent tumor saturation was seen.     

Radioimmunotherapy of human colon cancer xenografts by using 131I labeled-CAb1 F(ab')2

PURPOSE: Therapeutic efficacy, suitable dose, and administration times of 131I-CAb1 F(ab')2, a new monoclonal antibody therapeutics specifically directed against a cell surface-associated glycoprotein of colon cancer, were investigated in this article. METHODS AND MATERIALS: In human colon cancer xenografts, 131I-CAb1 F(ab')2 at the dose of 125 muCi, 375 muCi, and 1125 muCi were administrated intraperitoneally on Days 6 and 18 after implantation of HR8348 cells with CAb1 high reactivity. Survival time and tumor growth inhibition rate were used to evaluate the efficacy and safety of 131I-CAb1 F(ab')2 in treatment of colon cancer xenografts. RESULTS: Treatment of 125, 375, and 1125 muCi 131I-CAb1 F(ab')2 did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (p = 0.276, 0.865, 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 muCi and 1125 muCi 131I-CAb1 F(ab')2 were significantly longer than that of 5-FU-treated groups (p = 0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding 131I-labeled antibody dosage of 375 muCi and 1125 muCi. After single attack dosage, second reinforcement therapy may rise efficacy significantly. Tumor growth inhibition rates of 125 muCi, 375 muCi, and 1125 muCi 131I-labeled antibody on Day 20 posttherapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathology examination revealed that tissue necrosis of various degrees was found in 131I-CAb1 F(ab')2-treated groups. CONCLUSION: 131I-CAb1 F(ab')2 is safe and effective for colon cancer. It may be a novel and potentially adjuvant therapeutics for colon cancer.     

Therapeutic efficacy of anti-Lewis(y) humanized 3S193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with taxol chemotherapy.

Monoclonal antibody therapy may provide new treatment options in the management of metastatic breast cancer by selectively targeting tumors and producing a therapeutic effect, by delivering radiation or other toxins directly to tumor cells, or by producing an intrinsic immune inflammatory response. The effect of 131I-labeled humanized anti-Lewis(y) monoclonal antibody 3S193 (hu3S193) was compared with that of placebo and radiolabeled huA33 control antibody in a series of radioimmunotherapy experiments in a MCF-7 xenografted BALB/c nude mouse breast cancer model. The maximum tolerated dose of 131I-labeled antibody occurred at 200 microCi/mouse, at which dose level three of six mice that received 131I-hu3S193 showed significant tumor growth inhibition in contrast to no responses in the comparable 131I-huA33 control treatment arm. Breast cancer is an ideal model to test the efficacy of combined modalities given its known sensitivity to both radiotherapy and chemotherapy. The synergy between radioimmunotherapy and chemotherapy was therefore also explored using a combination of 131I-labeled hu3S193 antibody and Taxol using subtherapeutic doses of each agent. The combination of Taxol and 100 microCi of 131I-hu3S193 produced significant tumor inhibition in 80% of mice, whereas no responses were seen with either treatment modality alone or the combination of Taxol and 131I-huA33. These results support a potential therapeutic role of radiolabeled hu3S193 in the treatment of breast cancer, including combination therapy with Taxol, and warrants further investigation of this promising new agent.     

Targeting study of human hepatocellular carcinoma (HCC) using human HCC model in nude mice

Fifty-three nude mice bearing human HCC were used for targeting study of HCC using 131I-antihuman HCC isoferritin IgG. Of these mice, 17 were used for radioimaging with 131I-labeled IgG, 131I-labeled albumin and 131NaI. In labeled IgG group, all tumors were positively visualized by gamma camera with the best imaging on the 7th day after injection of labeled IgG (200 microCi, ip), the tumor/liver radioactivity ratio being 2.7. The dose of tumor radioactivity at the 7th day was 7-10 times higher than that in labeled albumin group. Thirty-six mice were used for the study of radioimmunotherapy with 131I-labeled IgG, 131NaI and IgG (n = 9, 300 microCi/50 micrograms, ip). The tumor inhibition rate in the labeled IgG group at 4th week after treatment was significantly higher than those of the other groups (81%, 60% and 18%, respectively, P less than 0.05). Tumor cell DNA analysis showed that the tumor cells were inhibited in S stage of the cell cycle. In the same way, five kinds of substances with affinity to HCC were studied in this animal model. The result indicates that the transplantable nude mice-human HCC model is acceptable for targeting study of HCC.     

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.     

Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's lymphoma xenografts.

Clinical trials of radioimmunotherapy (RIT) of lymphoma have produced frequent tumor regressions and remissions, but it has been difficult to determine to what extent these tumor responses have been due to antibody-specific targeted radiation, nontargeted radiation, and/or cytotoxicity mediated by the carrier monoclonal antibody (MoAb). In this report, RIT was studied in athymic nude mice bearing s.c. Raji human Burkitt's lymphoma xenografts using two different pan-B-cell MoAbs, MB-1 (anti-CD37) and anti-B1 (anti-CD20), which differ in isotype (and thus the potential for interaction with host effector mechanisms) and isotype-matched control antibodies either in the unlabeled state or labeled with 131I. When a single i.p. injection of 300 microCi 131I-labeled MB-1 (IgG1) was compared to treatment with unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 MoAb, an antibody-specific targeted radiation effect of RIT was seen. 131I-labeled MB-1 produced a 44 +/- 19% (SEM) reduction in tumor size at 3 weeks posttreatment, while unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 antibody treatment resulted in continued tumor growth over this period of time. In vitro studies demonstrated that MB-1 was incapable of mediating antibody-dependent cellular cytotoxicity using Raji tumor cell targets and human peripheral blood mononuclear cells. Similar to the MB-1 studies, treatment with 300 microCi 131I-labeled anti-B1 produced a 64% reduction in mean tumor size, while 300 microCi of control antibody resulted in a 58% increase in tumor size over the same 3-week period. In contrast to MB-1, however, unlabeled anti-B1 (an IgG2a MoAb which in vitro studies showed to be capable of antibody-dependent cellular cytotoxicity) also had a substantial antitumor effect. Indeed, 300 microCi 131I-labeled anti-B1 and unlabeled anti-B1 treatment (using an equivalent amount of total protein in the treatment dose) produced a similar specific reduction in tumor size. Increasing the radionuclide dose of anti-B1 to 450 microCi in another experiment did not produce a significant difference in tumor regression compared to a 300-microCi dose. These results suggest that the antitumor effects of 131I-labeled anti-B1 treatment were dominated by antibody-mediated cytotoxicity mechanisms, such that an antibody-specific targeted radiation effect could not be distinguished. In contrast, antibody-specific targeting of radiation was the dominant mechanism of tumor killing with 131I-labeled MB-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete tumor ablation with iodine 131-radiolabeled disialoganglioside GD2-specific monoclonal antibody against human neuroblastoma xenografted in nude mice

The antibody 3F8, an IgG3 murine monoclonal antibody (MoAb) against disialoganglioside GD2, could target iodine-131 (131I) to established subcutaneous human neuroblastoma (NB) xenografts in BALB/c nude mice. 131I-radiolabeled MoAb (0.125-1 mCi) was injected iv. Tumor radioactivity over time was calculated from scintigraphy, and radiation dose to individual tumors was calculated. Tumor shrinkage occurred only with 131I-labeled 3F8, but not with nonradioactive 3F8 or radiolabeled irrelevant antibody. While the tumor of the control mice enlarged by tenfold, the treated tumor showed over 95% shrinkage by 12 days. Both the rate of shrinkage and duration of tumor response were dose dependent. Calculated doses of more than 10,000 rad could be achieved. Only those tumors that received more than 4,200 rad were completely ablated without recurrence. Recurrent tumors were not antigen negative or radioresistant. These results confirmed the prediction based on imaging studies that human NB xenografts could be effectively eradicated with the use of 131I-labeled MoAb 3F8 with tolerable toxicities.     

Selective radiation of hematolymphoid tissue delivered by anti-CD45 antibody.

The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.     

Pharmacokinetics of 99mTc(Sn)- and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of 99mTc(Sn) and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant 125I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake [48.6% injected dose (ID)/g +/- 8.1% (SD)] and low tumor uptake (1.5% ID/g +/- 0.6%) for 99mTc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for 131I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the 131I-labeled fragments and poor tumor visualization with predominant kidney uptake for 99mTc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.     

Treatment of intracranial human glioma xenografts with 131I-labeled anti-tenascin monoclonal antibody 81C6

Lack of tumor specificity renders current modalities for treating malignant glioma ineffective. The administration of 131I-labeled monoclonal antibody (Mab) 81C6, which reacts with the glioma-associated extracellular matrix antigen, tenascin, to nude mice carrying s.c. human glioma xenografts has resulted in significant tumor growth delay and tumor regression. In this study, we evaluated the therapeutic efficacy of 131I-labeled 81C6 in athymic rats bearing intracranial human glioma xenografts, a more appropriate model for human gliomas. Mab 81C6, an IgG2b immunoglobulin, and an isotype-matched control Mab, 45.6, were labeled at 12.5-23.6 mCi/mg with chloramine-T. The Mabs were given i.v. at 1.25 and 2.5 mCi/animal for 131I-labeled 81C6, and 1.25 mCi for 131I-labeled 45.6 control. Therapeutic response was evaluated by survival prolongation using Wilcoxon rank sum analysis. Three experiments were done. No significant survival prolongation was found in the trial in which the average tumor size at the time of Mab administration was 60 +/- 14 mm3, two-thirds the size which causes animal death. In experiment 2, Mab was given at 16 +/- 14 mm3 average intracranial tumor volume. Statistically significant (P less than or equal to 0.005) survival prolongation was found for animals treated with 2.5 mCi 131I-labeled 81C6. In that experiment, male animals with intracranial xenografts had significantly shorter survival than females (P less than or equal to 0.005). When only female animals were used in the analysis, the 1.25-mCi 81C6 group also was found to have longer survival benefit (P less than or equal to 0.01). In the third experiment, only female animals were used and the tumor size at the initiation of treatment was 20 +/- 9 mm3. Highly significant survival prolongation again was found in both 1.25 (P = 0.001) and 2.5 mCi (P less than 0.001) 131I-labeled 81C6 groups. The estimated dose to intracranial tumors from 1.25 mCi of 131I-labeled Mab was 1585 rads for 81C6 and 168 rads for 45.6. Dose to other organs from 81C6 and 45.6 was similar, ranging between 31 rads to the brain and 734 rads to the bone marrow. However, normocellularity was observed in most marrow tissue examined microscopically. Three animals receiving the low dose (1.25 mCi 81C6) survived for more than 71 days with apparent cures. In conclusion, intracranial human glioma xenografts were treated successfully with 131I-labeled 81C6 but not control Mab.     

Hyperthermia enhances localization of 111In-labeled hapten to bifunctional antibody in human colon tumor xenografts

A unique bifunctional antibody (BFA) delivery system was examined for radiolocalization and distribution following hyperthermia (41.5 degrees C, 45 min) of T380h human colon tumor xenografts. The BFA is an F(ab')2 fragment made by combining two murine monoclonal antibodies with different specificities, one directed against carcinoembryonic antigen (monoclonal antibody CEM 231) and the other (monoclonal antibody CHA 255) against a hapten found on a derivative of 111In-labeled benzyl-EDTA (EOTUBE). This BFA is known as CEM/CHA. The CEM/CHA accumulates in carcinoembryonic antigen-expressing tissue and clears from normal tissues prior to administration of the radiolabeled hapten. T380h tumor chunks were injected s.c. into 31 nude mice. Two weeks later mean tumor volume was 352 mm3 and the animals were assigned to one of four groups: (a) CEM/CHA + hyperthermia + 111In-EOTUBE; (b) CHA 255 F(ab')2 + hyperthermia + 111In-EOTUBE, and (c and d) treated in the same manner as a and b, respectively, but without heat. The CEM/CHA, CHA 255 F(ab')2, and 111In-labeled hapten were injected i.p. at 14 micrograms, 7 micrograms, and 140-200 microCi/mouse, respectively. The hyperthermia was administered 22-24 h after BFA and the radiolabeled hapten was injected 2 h later. Twenty-four h thereafter, the animals were euthanized for testing. A significantly greater percentage of injected radioactivity localized within heated compared to unheated tumors in mice given CEM/CHA and 111In-EOTUBE (7.39%/g tumor and 4.46%/tumor versus 2.72%/g tumor and 1.44%/tumor, respectively). The percentage of kidney activity in mice given CHA 255 F(ab')2 fragments and heat was 57% lower than in the nonheated group when expressed on a per g basis (12.73 and 22.20%, respectively). Microautoradiography showed greater radiolocalization in heated tumors than in nonheated control tumors of comparable size. Semiquantification by immunoperoxidase staining for carcinoembryonic antigen did not reveal similar differences in the amounts of antigen present in tumors from heated and nonheated groups. These findings suggest that hyperthermia could be used to enhance delivery of radiolabeled haptens to prelocalized BFA and, thus, to enhance tumor imaging and therapeutic efficacy.