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Background: During metastasis, cancer cells migrate away from the primary tumour and invade the circulatory system and distal tissues. The stimulatory effect of growth factors has been implicated in the migration process. Placental growth factor (PlGF), expressed by 30–50% of primary breast cancers, stimulates measurable breast cancer cell motility in vitro within 3 h. This implies that PlGF activates intracellular signalling kinases and cytoskeletal remodelling necessary for cellular migration. The PlGF-mediated motility is prevented by an Flt-1-antagonising peptide, BP-1, and anti-PlGF antibody. The purpose of this study was to determine the intracellular effects of PlGF and the inhibiting peptide, BP-1. Methods: Anti-PlGF receptor (anti-Flt-1) antibody and inhibitors of intracellular kinases were used for analysis of PlGF-delivered intracellular signals that result in motility. The effects of PlGF and BP-1 on kinase activation, intermediate filament (IF) protein stability, and the actin cytoskeleton were determined by immunohistochemistry, cellular migration assays, and immunoblots. Results: Placental growth factor stimulated phosphorylation of extracellular-regulated kinase (ERK)1/2 (pERK) in breast cancer cell lines that also increased motility. In the presence of PlGF, BP-1 decreased cellular motility, reversed ERK1/2 phosphorylation, and decreased nuclear and peripheral pERK1/2. ERK1/2 kinases are associated with rearrangements of the actin and IF components of the cellular cytoskeleton. The PlGF caused rearrangements of the actin cytoskeleton, which were blocked by BP-1. The PlGF also stabilised cytokeratin 19 and vimentin expression in MDA-MB-231 human breast cancer cells in the absence of de novo transcription and translation. Conclusions: The PlGF activates ERK1/2 kinases, which are associated with cellular motility, in breast cancer cells. Several of these activating events are blocked by BP-1, which may explain its anti-tumour activity.

Radioimmunotherapy (RAIT) has been shown to enhance the efficacy of chemotherapy in a variety of tumor models. When multiple drugs are available for a given tumor type, several questions arise, such as how one selects the appropriate drug to be used with RAIT, in what sequence to administer the two therapeutic agents and how to space the two modalities. Unique molecular characteristics affecting chemo- and radiosensitivity are found in every tumor. We postulated that the agents and dose scheduling of bimodal RAIT plus chemotherapy might have to be tailored to each tumor based on the expression of specific relevant genes.To determine whether a single choice of agents and sequence and spacing of agents would be effective in 4 established human ovarian cancer cell lines, the cytotoxic effect of four standard first- and second-line chemotherapeutic agents used to treat ovarian cancer (doxorubicin, carboplatin, paclitaxel, or topotecan) or radioimmunotherapy (90Y-RS-7 IgG anti-EGP1) were evaluated as single agents or as a bimodal therapy. The four human ovarian cancer lines differed with respect to expression of p53 and drug resistance proteins: SKOV3 (p53null, mdr-, mrp+), A2780 (p53wt, mdr+, mrp), IGROV-1 (p53mut, mdr, mrp+), and OVCAR3 (p53mut, mdr-, mrp-).The profile of chemo- and radiosensitivity for the 4 drugs and for RAIT was unique for each of the four tumor lines. As a result, combinations of radio-antibody and chemotherapy that resulted in good growth inhibition in one tumor line were ineffective in another. Several specific synergistic and antagonistic combinations were identified for each of the ovarian cancer cell lines studied.These studies provide evidence that individualized bimodal RAIT and chemotherapy has to be used for each tumor. Therefore, the influence of individual molecular traits on growth inhibition effects from different combinations of agents needs to be studied. This work should then permit rational choices of which drug to add to RAIT, which dose-schedule to use (sequential with RAIT first or sequential with drug first) and how to space the two modalities, based on certain phenotypic markers of the tumors.

Inhibition of the enzyme cyclooxygenase with celecoxib is cytotoxic in a variety of solid tumor cell lines. Previous work has shown that by charting circadian rhythms, it has been possible to find optimal times to deliver a dose of drug, such that it is most efficacious in killing cancer cells and least harmful to normal tissues. Therefore, we examined the time dependence of toxicity (chronotoxicity) and of antitumor effects (chronotherapy) of celecoxib to determine optimal time of day for dosing with respect to light-dark cycles.Celecoxib was administered i.p. for 10 days (5 days on, 2 days off, 5 days on) to nude mice bearing s.c. breast xenografts. Body weight, peripheral blood cells, clinical chemistry, and tumor growth were monitored.The highest tolerance (100% survival) was found at 7 HALO and the least occurred at 17 h after light onset (HALO; 10% survival). Chronotherapy at a 20-mg/kg dose varied between the seven HALO evaluated and between the three breast tumors (MCF-7, ZR-75-30, and MDA-MB-468) studied. When the maximum tolerated dose (MTD) of celecoxib was optimized for each HALO, we found that at 7-10 HALO, the MTD was 25 mg/kg, whereas at 17-20 HALO; the MTD was only 10 mg/kg. Tumor regression was observed when dosing was done at 23 HALO to 7 HALO (5 a.m. to 1 p.m.), whereas no therapeutic response was observed when dosing was done at 10-13 HALO (4 p.m. to 7 p.m.), and rapid tumor growth was noted when dosing was done at 17 HALO (11 p.m.).Tumor growth response to the MTD at each HALO revealed that there was no clear relationship between dose administered and therapeutic response. COX-2 expression was not able to explain either the chronotherapy or the chronotoxicity results obtained.

A radiolabeled antibody (Ab) to CD74 (the MHC class II invariant chain, Ii) was shown previously to effectively kill human B-lymphoma cells in vitro. Conjugates with both Auger electron and beta-particle emitters were able to kill cells, but the former displayed less nonspecific toxicity in the in vitro assay used. In this report, we have extended the studies to an in vivo model of tumor growth. The human B-cell lymphoma Raji was injected i.v. into severe combined immunodeficient mice, and radiolabeled Abs were injected at various times after tumor inoculation. The maximum tolerated dose (MTD), as well as lower doses, was tested. Tumor growth was monitored by hind-leg paralysis. With a 3-5-day interval before Ab injection, anti-CD74 conjugated to either (111)In or (67)Ga, at a dose of 240-350 microCi/mouse, produced a strong therapeutic effect, with greatly delayed tumor growth, and many of the treated mice were tumor free for6 months. Control mice became paralyzed in 16-24 days, uniformly. Treatment at later time points (9-day interval) had little therapeutic effect. The MTD was required for optimal therapy. With the beta-particle emitter (90)Y, the MTD was much less, 25 microCi/mouse, and at this dose there was only a weak therapeutic effect. In conclusion, the data suggest that low-energy electrons are more effective than beta-particles in this model system. These results may be applicable to humans, particularly in the case of micrometastatic disease. This approach may also be effective with other Abs that accrete in large amounts.

Tumor targeting and therapeutic efficacy of (177)Lu-labeled monoclonal antibody (mAb) RS7 (antiepithelial glycoprotein-1) was evaluated in a human nonsmall cell lung carcinoma xenograft model. The potential of (177)Lu-labeled RS7 was compared with that of RS7 labeled with (90)Y and a residualizing form of (131)I.A 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) conjugate of RS7 was used for radiolabeling with (177)Lu-acetate or (88/90)Y-acetate. Biodistribution and therapy studies were conducted in nude mice with subcutaneous Calu-3 xenografts. Therapy studies were performed using the maximal tolerated doses (MTDs) of (90)Y-DOTA-RS7 (3.9 MBq [105 microCi]) and (177)Lu-DOTA-RS7 (10.2 MBq [275 microCi]) and compared with the data obtained using the MTD (13.0 MBq [350 microCi]) of a residualizing form of (131)I-RS7.Radiolabeling of RS7-DOTA conjugate with (177)Lu-acetate was facile. (177)Lu-DOTA-RS7 displayed biodistribution results that were nearly identical to that of the (88)Y analog in a paired-label study. The mean percentage injected doses per gram (%ID/g) for (177)Lu-RS7 and (88)Y-RS7 (in parentheses) in tumor were 38.3 %ID/g (39.1 %ID/g), 63.0 %ID/g (66.0 %ID/g), 63.0 %ID/g (65.8 %ID/g), and 34.0 %ID/g (34.9 %ID/g) on days 1, 3, 7, and 14, respectively. Elimination of established tumors, with an initial mean tumor volume of 0.24 cm(3), was shown using doses of (177)Lu-DOTA-RS7 ranging from 5.6 to 9.3 MBq (150--250 microCi) per nude mouse, with no significant difference in response rate noted between the doses in this range. Specificity of the therapeutic effect was shown in an isotype-matched control experiment, in which (177)Lu-DOTA-RS7 was markedly more effective than the (177)Lu-DOTA control antibody. A comparison of the therapeutic efficacies of (177)Lu-DOTA-RS7 and (90)Y-DOTA-RS7, using mice with established tumors with an initial mean tumor volume of 0.85 cm(3), indicated similar tumor growth inhibition and similar tumor regrowth profiles. The therapy data were similar to those obtained with residualizing (131)I-RS7 obtained at the same time.(177)Lu-RS7 is an effective radioimmunoconjugate for radioimmunotherapy. With its radiophysical properties similar to those of (131)I, coupled with its facile and stable attachment to mAb, (177)Lu promises to be an alternative to (131)I, and a complement to (90)Y, in radioimmunotherapy.

In pivotal phase III clinical trials for detecting recurrent or metastatic colorectal cancer, most patients received a single arcitumomab injection. However, the early detection of postsurgical recurrence or metastases with arcitumomab will necessitate serial studies for surveillance. We present immunogenicity, safety, and imaging data supporting the use of multiple administrations of arcitumomab.Human antimouse antibody (HAMA) response, adverse events, clinical laboratory values, and diagnostic imaging results were evaluated in 44 patients (24 men, 20 women; age range, 2878 y) after repeated arcitumomab administration (44 second and 3 third injections). Most patients initially had Dukes' class B or C colorectal cancer and had known or occult disease recurrence and elevated serum carcinoembryonic antigen levels at the time of the repeated injection.At the repeated injection, in no patient did elevated HAMA titers develop, hematology and serum chemistry changes were clinically insignificant, and only 1 adverse event (eosinophilia) was judged at least possibly related to arcitumomab. Arcitumomab imaging results at the second injection were comparable with those obtained in phase III trials after a single injection of arcitumomab, having a 78% per-lesion concordance with CT in the abdomen and pelvis and a 73% sensitivity and 94% specificity based on 9 patients with cancer confirmed surgically at 11 anatomic sites and excluded at 16 sites.These data indicate that at least 2 injections of arcitumomab can be given safely to patients with colorectal cancer, without increased immunogenicity and with imaging efficacy equivalent to the first administration.

This phase I study was initiated to determine the toxicity and therapeutic potential of high-dose 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen monoclonal antibody (MAb) combined with autologous hematopoietic stem cell rescue (AHSCR) in patients with rapidly progressing metastatic medullary thyroid cancer.Twelve patients were entered into the study. Dose escalation was based on prescribed radiation doses to critical organs (i.e., kidney, lung, and liver). Starting doses were 900 cGy to the kidney and no more than 1200 cGy to the lung and liver, with dose increments of 300 cGy until the maximum tolerable dose is determined. Tumor targeting was assessed by external scintigraphy, toxicity was assessed according to the common toxicity criteria of the National Cancer Institute, and therapy responses were assessed by CT, serum carcinoembryonic antigen, and calcitonin.One patient received 9.95 GBq 131I-MN-14 F(ab)2, for an initial dose of 656 cGy to critical organs, 8 received 900 cGy (8.69-17.98 GBq), and 3 received 1200 cGy (15.17-17.69 GBq). The MAb scans of all patients showed positive findings. Autologous hematopoietic stem cells were given to all patients 1-2 wk after therapy, when the total body radiation exposure was less than 5.2 x 10(-7) C/kg/h. Dose-limiting toxicity, defined as grade 3 or 4 nonhematologic toxicity, was not seen in the patient who received the 656-cGy dose, and only 1 of the 8 patients treated at the 900-cGy dose level had grade 3 toxicity, which was gastrointestinal and reversible. No dose-limiting toxicity was seen in the 3 patients treated at the 1200-cGy dose level. Except for the instance of grade 3 gastrointestinal toxicity, nonhematologic toxicity was relatively mild, with only grade 1 or 2 toxicity observed in 9 patients. No renal toxicity was seen. Of the 12 patients, 1 had partial remission for 1 y, another had a minor response for 3 mo, and 10 had stabilization of disease lasting between 1 and 16 months.The results show the safety of administering high myeloablative doses of 131I-MN-14 F(ab)2 with AHSCR in patients with metastatic medullary thyroid cancer. The antitumor responses in patients with aggressive, rapidly progressing disease are encouraging and warrant further research to optimize the effectiveness of this new treatment.

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacetic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1- and 125I-IMP-R2-labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, (111)In, and 131I-dilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 125I-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 +/- 4.1% and 18.1 +/- 7.9% (P0.05) for the conventional 131I- and 125I-IMP-R1-RS7, respectively, and tumor:nontumor ratios were 2.6-4.5-fold higher with the 125I-IMP-R1-RS7. It is estimated that 131I-IMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 90Y-labeled RS7, and 0.7 times that of 131I-DLT-labeled RS7. Tumor accretion of 125I-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor:nontumor ratios were observed when 125I-IMP-R1-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way.

Usually, the red marrow (RM) is the first dose-limiting organ in radioimmunotherapy. However, several studies have obtained only poor correlations between the marrow doses and the resulting toxicities. Furthermore, RM doses are mostly not determined directly but are derived from blood doses by assuming a ratio that is, over time for the respective conjugates, more or less constant between blood and marrow activities. The aim of this study was to determine, in a mouse model, this RM:blood activity ratio for various immunoconjugates, to investigate whether there may be differences between complete IgG and its fragments with various labels ((125/131)I versus (111)In, (88/90)Y, or 213Bi), and to analyze, in more detail, factors other than just total dose, such as dose rate or relative biological effectiveness factors, that may influence the resulting myelotoxicity. The maximum tolerated activities (MTAs) and doses (MTDs) of several murine, chimeric, and humanized immunoconjugates as complete IgG or fragments (F(ab)2 and Fab), labeled with beta(-)-emitters (such as 131I or 90Y), Auger electron-emitters (such as 125I or (111)In), or alpha-emitters (such as 213Bi) were determined in nude mice. Blood counts were monitored at weekly intervals; bone marrow transplantation was performed to support the assumption of the RM as dose-limiting. The radiation dosimetry was derived from biodistribution data of the various conjugates, accounting for cross-organ radiation; besides the major organs, the activities in the blood and bone marrow (and bone) were determined over time. Whereas no significant differences were found for the RM:blood ratios between various IgG subtypes, different radiolabels or various time points, differences were found between IgG and bi- or monovalent fragments: typically, the RM:blood ratios were approximately 0.4 for IgG, 0.8 for F(ab')2, and 1.0 for Fab'. Nevertheless, at the respective MTAs, the RM doses differed significantly between the three conjugates: e.g., with 131I-labeled conjugates, the maximum tolerated activities were 260 microCi for IgG, 1200 microCi for F(ab)2, and 3 mCi for Fab, corresponding to blood doses of 17, 9, and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG, and still 3 times higher as compared to F(ab)2; interestingly, all three deliver approximately 4 Gy within the first 24 h. The MTDs of all three conjugates were increased by BMT by approximately 30%. Similar observations were made for 90Y-conjugates. Higher RM doses were tolerated with Auger-emitters than with conventional beta(-)-emitters, whereas the MTDs were similar between alpha- and beta(-)-emitters. In accordance to dose rates never exceeding those occurring at the single injection MTA, two subsequent injections of two doses of 80% of the single shot MTA of 131I- or 90Y-labeled Fab' and two doses of 100% of the single shot MTA of 213Bi-labeled Fab' were tolerated without increased lethality, if administered 24-48 h apart. In contrast, reinjection of bivalent conjugates was not possible within 6 weeks. These data suggest that the RM:blood activity ratios differ between IgG and fragments, although there is no anatomical or physiological explanation for this phenomenon at this point. In contrast to the current opinion, indication for a strong influence of the dose rate (or dose per unit time), not only total dose, on the resulting toxicity is provided, whereas the influence of high-linear energy transfer (alpha and Auger/conversion electrons) versus low-linear energy transfer (beta and gamma) type radiation seems to be much lower than expected from previous in vitro data. The lower myelotoxicity of Auger-emitters is probably due to the short path length of their low-energy electrons, which cannot reach the nuclear DNA if the antibody is not internalized into the stem cells of the RM.

Antibody (Ab) fragments are preferred agents for imaging applications because of their rapid clearance from the blood, thereby providing high tumor:blood ratios within a few hours. Several preclinical studies have also suggested that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fragments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a deltaCH2 (deletion mutant missing the CH2), a gamma3 F(ab')2 containing the human IgG3 hinge, and three glycosylated variants. The gamma3 F(ab')2 and glycosylated variants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma3 F(ab')2 variant contains 10 cysteine residues that could be used for direct coupling using thiol chemistry, whereas the glycosylated variants have N-linked glycosylation sites engineered in the CH1 domain (two variants) as well as the VK domain (one variant). All of these variants were successfully prepared and shown to react with the target antigen. All Abs could be purified to a single peak by size-exclusion HPLC, but the deltaCH2 variant showed two distinct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in the extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the carbohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mice. All of the humanized Ab divalent fragments cleared nearly 20 times faster from the blood than the murine parent F(ab')2 over a 24-h period. The glycosylated fragments showed some added stability compared to the other fragments over 4 h, but by 24 h, they had cleared to the same extent. Size-exclusion high-performance liquid chromatography of blood samples indicated that the humanized Ab fragments were quickly degraded in the blood. Thus, there is an inherent instability of the divalent fragments from these humanized IgG1 constructs that may affect their utility in imaging or therapy applications.

We have shown previously significant antitumor effects using 90Y-MN-14 anti-CEA monoclonal antibody (MAb) for radioimmunotherapy (RAIT) of human medullary thyroid cancer (MTC) xenografts using the TT cell line. The purpose of this investigation was to determine the effect of combining chemotherapy and RAIT with 90Y-MN-14 in MTC. In particular, the toxicity and efficacy of various dose schedules of RAIT and doxorubicin were examined and compared with that at the maximum tolerated dose (MTD) of each single modality treatment. The MTD of RAIT of 105 microCi of 90Y-MN-14 was given alone and combined with 100 and 75% of the MTD of doxorubicin (60 mg/m2); and the MTD of doxorubicin was given alone and combined with 100 and 75% of the MTD of RAIT. In addition, 75% of each agent was also administered in combination. The MTD of RAIT was also evaluated in combination with 58 and 78% of the MTD of Taxol. Whereas 90Y-MN-14 (105 microCi) led to significant antitumor effects (P0.0001), doxorubicin at 60 mg/m2 or Taxol at 225 mg/m2 yielded only a slight tumor growth delay. The combinations of 100% of the MTD of RAIT and 75% of the MTD of doxorubicin and 100% of the MTD of doxorubicin and 75% of the MTD of RAIT were equitoxic to the MTD of RAIT alone and appear to result in improved efficacy compared with either RAIT or doxorubicin alone. For the 100% RAIT and 75% doxorubicin combination, the therapeutic efficacy was similar when doxorubicin was administered on the same day or 1 day after RAIT, but the treatment was less effective when doxorubicin was administered 2 days after RAIT (P0.03). Prolonged retardation of tumor progression was also observed in animals treated with the MTD of RAIT combined with 175 mg/m2 of Taxol, without increases in toxicity above that observed with RAIT alone. In conclusion, the combination of RAIT and chemotherapy appears to augment the antitumor effects of either treatment alone without a significant increase in toxicity. In addition, the timing of drug administration relative to RAIT in the combined therapy appears to be important.

Both CD22 and CD20 have been used successfully as target molecules for radioimmunotherapy (RAIT) of low-grade B cell non-Hodgkin's lymphoma. Because both CD20 and CD22 are highly expressed relatively early in the course of B cell maturation, and because its expression is maintained up to relatively mature stages, we studied the potential of the humanized anti-CD22 antibody, hLL2, as well as of the chimeric anti-CD20 (chCD20) antibody, rituximab (IDEC-C2B8), for low- or high-dose (myeloablative) RAIT of a broad range of B cell-associated hematological malignancies. A total of 10 patients with chemorefractory malignant neoplasms of B cell origin were studied with diagnostic (n = 5) and/or potentially therapeutic doses (n = 9) of hLL2 (n = 4; 0.5 mg/kg, 8-315 mCi of 131I) or chCD20 (n = 5; 2.5 mg/kg, 15-495 mCi of 131I). The diagnostic doses were given to establish the patients' eligibility for RAIT and to estimate the individual radiation dosimetry. One patient suffered of Waldenström's macroglobulinemia, eight patients had low- (n = 4), intermediate- (n = 2) or high- (n = 2) grade non-Hodgkin's lymphoma, and one patient had a chemorefractory acute lymphatic leukemia, after having failed five heterologous bone marrow or stem cell transplantations. Three of these 10 patients were scheduled for treatment with conventional (30-63 mCi, cumulated doses of up to 90 mCi of 131I) and 7 with potentially myeloablative (225-495 mCi of 131I) activities of 131I-labeled hLL2 or chCD20 (0.5 and 2.5 mg/kg, respectively); homologous (n = 6) or heterologous (n = 1) stem cell support was provided in these cases. Good tumor targeting was observed in all diagnostic as well as posttherapeutic scans of all patients. In myeloablative therapies, the therapeutic activities were calculated based on the diagnostic radiation dosimetry, aiming at lung and kidney dosesor = 20 Gy. Stem cells were reinfused when the whole-body activity retention fell below 20 mCi. In eight assessable patients, five had complete remissions, two experienced partial remissions (corresponding to an overall response rate of 87%), and one (low-dose) patient had progressive disease despite therapy. In the five assessable, actually stem-cell grafted patients, the complete response rate was 100%. Both CD20 and CD22 seem to be suitable target molecules for high-dose RAIT in a broad spectrum of hematological malignancies of B cell origin with a broad range of maturation stages (from acute lymphatic leukemia to Waldenström's macroglobulinemia). The better therapeutic outcome of patients undergoing high-dose, myeloablative RAIT favors this treatment concept over conventional, low-dose regimens.

Nine radioimmunotherapy (RAIT)-naive patients with medullary thyroid cancer received high doses of 131I-MN-14 F(ab)2 anti-carcinoembryonic antigen monoclonal antibody (232-486 mCi), five in combination with bone marrow harvest, without prior granulocyte colony stimulating factor (G-CSF) injections (group 1) and the other four using peripheral blood stem cell harvest (PBSCH) preceded by G-CSF administration of 10 microg/kg per day for 5 days for stem cell mobilization, 6-8 days before RAIT (group 2). The amounts of radioactivity (mCi) given in both groups were similar (312 +/- 93 versus 424 +/- 65; P = NS). The percent platelet loss at nadir, duration of grade 4 thrombocytopenia, and time to complete recovery (TTCR, measured from the day of treatment), were 83 +/- 17%, 2.5 +/- 0.7 days, and 45 +/- 8 days in group 1, respectively, compared with 88 +/- 6%, 3.0 +/- 2.6 days, and 50 +/- 24 days in group 2 (P = NS), respectively. In contrast, the percent WBC loss at nadir, duration of grade 4 leukopenia, and TTCR of WBCs were 72 +/- 12%, 0.0 +/- 0.0 day, and 42 +/- 12 days in group 1, respectively, compared with 93 +/- 3%, 8.0 +/- 3.6 days, and 263 +/- 136 days in group 2, respectively (P0.02, 0.03, and 0.05 for differences of percent loss, duration of nadir, and TTCR, respectively). The difference in WBC toxicity after RAIT with bone marrow harvest and PBSCH is thought to be due to the administration of G-CSF for stem cell mobilization within 1 week before RAIT, which may sensitize the "endogenous" granulocyte precursors to subsequent RAIT. Preclinical data of RAIT in mice showed that the time of G-CSF administration before RAIT is critical: increased WBC toxicity was seen in mice given RAIT 3 or 7 days after a 5-day course of G-CSF (81 and 57% WBC loss, respectively) compared with those given no G-CSF or G-CSF 10 or 14 days before RAIT (45-50%) WBC loss). In conclusion, our data indicate that the timing of RAIT after the administration of G-CSF for PBSCH may influence WBC toxicity and recovery after this treatment and may have important implications on the design of high-dose RAIT trials combined with PBSCH.

At the time of surgery, occult metastases (micrometastases) are present in more than 50% of colorectal cancer patients, and the liver is the most frequent site of apparent metastatic disease. Frequently, adjuvant chemotherapy is unable to prevent tumor recurrence. Thus, novel therapeutic strategies are warranted. The aim of this study was to establish a model of human colon cancer metastatic to the liver of nude mice, to assess, in this setting, the therapeutic efficacy of radioimmunotherapy (RAIT) compared to standard chemotherapy and to evaluate, in a Phase I/II trial, the toxicity and therapeutic efficacy of RAIT in colorectal cancer patients with small volume disease metastatic to the liver. Multiple liver metastases of the human colon cancer cell line GW-39 were induced by intrasplenic injection of a 10% tumor cell suspension. Whereas controls were left untreated, therapy was initiated on day 10 or 20 after tumor inoculation with the 131I-labeled, low affinity anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAb), F023C5 (Ka = 10(7) liters/mol), or the high-affinity anti-CEA MAb, MN-14 (Ka = 10(9) liters/mol), or chemotherapy (5-fluorouracil/leucovorin (folinic acid) versus irinotecan) at their respective maximum tolerated doses (MTDs). Twelve colorectal cancer patients with small volume disease metastatic to the liver (all lesionsor = 2.5 cm) were entered into a mCi/m2-based Phase I dose escalation study with 131I-labeled humanized version of MN-14, hMN-14. The patients were given single injections, starting at 50 mCi/m2 and escalating in 10-mCi/m2 increments. The MTD was defined as the dose level at whichor = 1 of 6 patients develop grade 4 myelotoxicity. In the mice, untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation. The life span of mice treated with 5-fluorouracil/leucovorin was prolonged for only 1-3 weeks, whereas irinotecan led to a 5-8-week prolongation. In contrast, at their respective MTDs, the 131I-labeled low-affinity anti-CEA MAb, F023C5, led to a 20% permanent cure rate, and the high affinity MAb, MN-14, led to an 80% permanent cure rate, when therapy was initiated at 10 days after tumor inoculation. In the 20-day-old tumor stage, although it prolonged life, 131I-F023C5 was unable to achieve cures, whereas 131I-MN-14 was still successful in 20%. Histologically, no remaining viable tumor cells could be demonstrated in these animals surviving6 months. In patients, the MTD was reached at 60 mCi/m2 of hMN-14 (at 70 mCi/m2, two of three grade 4 myelotoxicities). Of 11 assessable patients, 2 had partial remissions (corresponding to an objective response rate of 18%), and 5 (45%) had minor/mixed responses or experienced stabilization of previously rapidly progressing disease. These data suggest that in small volume disease, RAIT may be superior to conventional chemotherapy. Antibodies of higher affinity seem to be clearly superior. The clinical response rates in patients with small volume disease are encouraging, being comparable to the response rates of conventional chemotherapeutic regimens but with fewer side effects. Ongoing studies will show whether treatment at the MTD will further improve therapeutic results.

This study was undertaken to determine the factors affecting myelotoxicity after radioimmunotherapy (RAIT) with 131I-labeled anticarcinoembryonic antigen (anti-CEA) monoclonal antibodies (MAbs).Ninety-nine patients who received 131I-labeled MN-14 or NP-4 anti-CEA MAbs for the treatment of CEA-producing cancers were assessed for platelet and white blood cell (WBC) toxicity based on the common Radiation Therapy Oncology Group (RTOG) criteria. Univariate and multivariate regression analyses were used to identify the statistically significant factors affecting toxicity among the following variables: red marrow dose, baseline platelet and WBC counts, bone or marrow (or both) metastases, prior chemo- or radiotherapy, timing of prior chemo- or radiotherapy in relation to RAIT, type and number of prior chemotherapeutic regimens, age, sex, antibody form and cancer type.Red marrow dose, baseline platelet or WBC counts and multiple bone or marrow (or both) metastases were the only significant factors affecting hematologic toxicity according to both univariate and multivariate analyses, whereas chemotherapy, 3-6 mo before RAIT, was significant according to multivariate analysis. In this retrospective study, the multivariate regression equations using these four variables provided an exact fit for postRAIT platelet toxicity grade (PltGr) and WBC toxicity grade (WBCGr) in 40% and 46%, respectively, of the 99 patients included in the analysis. Moreover, severe (grade 3 or 4) PltGr and WBCGr could be classified accurately in all cases, whereas nonsevere (grade 0, 1, or 2) PltGr and WBCGr could be classified accurately in all but 6 of 13 cases of grade 2 toxicity, in which a severe toxicity grade was estimated using the regression equations.Red marrow dose, baseline blood counts, multiple bone or marrow (or both) metastases and recent chemotherapy are the most important factors related to hematologic toxicity after RAIT. This study provides a simple model for predicting myelotoxicity with reasonable accuracy in most patients. In addition, the identification of bone or marrow (or both) metastases and recent chemotherapy as significant factors for myelotoxicity may be important in the future design of clinical trials.

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 asor = 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 by3-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 of35 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.

Monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) have been recognized as targeting agents for medullary thyroid carcinoma (MTC). This Phase I/II study was initiated to determine the safety, maximum tolerated dose (MTD), and therapeutic potential of (131)I-MN-14 F(ab)2 anti-CEA MAb for patients with metastatic MTC.Fifteen patients were enrolled in this study. Dose escalation was based on estimates of radiation dose to the bone marrow, and the radioactive dose given was determined by a pretherapy diagnostic study in which 8 mCi (0.6-20 mg) of (131)I-MN-14 F(ab)2 was administered 1 week prior to therapy.Three patients received an initial dose of 140 centigray (cGy) to bone marrow, 11 received 180 cGy, and 1 received 220 cGy. Myelosuppression was the only significant treatment-related dose-limiting toxicity (DLT), and the MTD appeared to be 180 cGy to the bone marrow. Human antimouse antibodies (HAMA) developed in 8 patients 2-6 weeks after therapy. Seven patients had a median of 55% reduction of tumor markers. One patient showed a dramatic improvement in the mass effect on the airways caused by 3 tumor lesions in the neck, with a 45% reduction of overall tumor burden. The disease has continued to be radiologically stable in 11 of 12 assessable patients for periods ranging from 3+ to 26+ months.Therapy with (131)I-MN-14 F(ab)2 is well tolerated and shows evidence of biochemical and radiologic antitumor activity. HAMA development suggests that humanized MAbs will be required in trials with repeated dose schedules. Further dose escalation, alone or in combination with other therapy modalities, is indicated for future trials, preferably with humanized anti-CEA MAbs.

The purpose of this study was to evaluate the safety and efficacy of a technetium 99m-labeled antigranulocyte antibody Fab' fragment (sulesomab) as a diagnostic imaging agent in patients with suspected acute, nonclassic appendicitis.This prospective multicenter trial involved 141 children and adults with suspected acute, nonclassic appendicitis. The investigators interpreted planar images acquired 15 to 30 minutes and 1, 2, and 4 hours after injection and also by single-photon emission computed tomography (SPECT). The imaging results were confirmed surgically, whereas nonsurgical patients were considered as not having appendicitis (intent to treat).Sulesomab had a sensitivity of 91% (29/32 patients) and a negative predictive rate of 97% for acute appendicitis. It detected additional abnormalities in 7 of 9 patients with other inflammatory abdominal disease and had a specificity of 92% (91/99 patients) and a positive predictive value of 80% for surgically confirmed right-lower-quadrant disease. In positive studies, 26% were identified by planar imaging at 15 to 30 minutes, 46% by 1 hour, 63% by 2 hours, and 71% by 4 hours; 29% required SPECT to detect the abnormality. Scanning time was 5 to 10 minutes per planar image and about 45 minutes for a SPECT study. Investigators found that sulesomab would have changed clinical management or reduced additional diagnostic studies in 64% of the patients. Adverse events were infrequent, minor, and self-limiting (9/141 patients, 6%). No human antimurine antibody response occurred in 48 evaluable patients.Sulesomab is safe, well-tolerated, and with no apparent immunogenicity. Focal inflammation or infection in the setting of suspected atypical appendicitis is rapidly and accurately detected. Management decisions incorporating sulesomab imaging potentially provide clear patient benefits, especially by correctly predicting surgery to be unnecessary in 97% of patients without acute appendicitis.

Myelosuppression is the dose-limiting side effect of most forms of radioimmunotherapy (RAIT). Long-term leukopenia (4-8 weeks) has been documented from a single maximum tolerated dose (MTD) in experimental mice. Several methods for alleviating RAIT-induced marrow toxicity have been evaluated preclinically, including cytokine intervention, bone marrow transplantation (BMT), and hemoregulatory peptide administration. To improve the therapeutic potential of RAIT, multiple doses of radioantibody must be delivered. Maximizing the frequency of radioantibody administration is desirable. However, little is known about the myelotoxic effects of multiple cycles of RAIT. In the studies presented here we compared the magnitude of myelosuppression, the time of nadir, and the duration of toxicity associated with one or two MTDs of 1-131-MN-14 anti-carcinoembryonic antigen immunoglobulin G (250 microCi) administered to BALB/c mice 49 days apart, the shortest interval possible without producing lethality. Studies were conducted with radioantibody alone or with cytokines (interleukin-1/granulocyte-macrophage colony-stimulating factor), BMT, or Hp5b to determine whether bone marrow became more "brittle" after the first dose. Profiles of myelosuppression and recovery were monitored weekly for 7 weeks after each dose in both granulocyte and lymphocyte populations. The results demonstrated that granulocyte suppression was greater and of longer duration after the second dose of RAIT administered alone, with cytokines, or with BMT, but less severe with Hp5b. For example, in the RAIT + BMT treatment, the second dose resulted in an 87% loss of granulocytes, whereas a 30% loss occurred after the first dose. The nadir of toxicity lasted until days 21 to 28 after the second dose and until day 14 after the first dose. Lymphocyte suppression was of greater duration after the second cycle of RAIT alone or RAIT with BMT, plateauing at50% of untreated levels between days 28 and 49, but was of shorter duration when RAIT was given with cytokines or Hp5b. The results are discussed in terms of 1) the radiosensitivity of each subpopulation, 2) the effects on progenitors and on stromal cells, 3) the benefits of increasing dose frequency vs. increasing dose intensity, and 4) the possibility of using preclinical data to optimize the frequency of dosing in patient trials.

Whereas bivalent fragments have been widely used for radio-immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of (131)I-labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose-limiting organ toxicity and anti-tumor efficacy. The maximum tolerated doses (MTDs) and dose-limiting organ toxicities of the (131)I-labeled anti-CEA antibody MN-14 [IgG, F(ab')2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D-lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 (131)I-labeled immunoconjugates [IgG, F(ab')2 and Fab], the red marrow was the only dose-limiting organ; MTDs were 260 microCi for IgG, 1,200 microCi for F(ab')2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab')2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti-tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments.

Recent clinical results suggest that higher anti-tumor efficacy may be achieved with internalizing monoclonal antibodies (MAbs) at lower toxicity when labeled with Auger-electron, as compared to conventional beta-emitters. The aim of our study was to compare the toxicity and anti-tumor efficacy of the 125I-labeled internalizing MAb, CO17-1A, with its 131I-labeled form in a human colon cancer model in nude mice. Biodistribution studies were performed in nude mice bearing s.c. human colon cancer xenografts. For therapy, the mice were injected either with unlabeled 125I- or 131I-labeled C017-1A at equitoxic doses. Control groups were left untreated, were given a radiolabeled isotype-matched irrelevant antibody or a tumor-specific, but noninternalizing antibody. The maximum tolerated activities (MTD) of 131I-and 125I-CO17-1A without artificial support were 300 microCi and 3 mCi, respectively. Myelotoxicity was dose-limiting; bone marrow transplantation allowed for an increase of the MTD to 400 microCi of 131I-17-1A, whereas the MTD of 125I-17-1A with bone marrow support had not been reached at 5 mCi. Whereas no significant therapeutic effects were seen with unlabeled C017-1A, tumor growth was retarded with 131I-CO17-1A. With the 125I-label, however, therapeutic results were clearly superior. In contrast, no significant difference was observed in the therapeutic efficacy of the 131I- vs. 125I-labeled, noninternalizing antibodies. Our data indicate a superiority of Auger-electron emitters, such as 125I, as compared to therapy with conventional beta-emitters with internalizing antibodies. The lower toxicity of Auger emitters may be due to the short path length of their low-energy electrons, which can reach the nuclear DNA only if the antibody is internalized (as is the case in antigen-expressing tumor tissue, but not in the stem cells of the red marrow).

The biodistribution, pharmacokinetics and dosimetry of 188Re-labeled MN-14, an IgG anti-carcinoembryonic antigen monoclonal antibody (MAb), were assessed in patients in advanced gastrointestinal cancer. In addition, the dose-limiting toxicity (DLT) and maximum tolerated dose of fractionated doses of this agent were determined.Eleven patients were administered radioactive doses of directly labeled 188Re-MN-14 IgG, ranging from 20.5 mCi to 161.0 mCi (2.0 mg-4.9 mg). Ten of these patients received two or three MAb infusions, given 3-4 days apart, delivering total doses of 30 mCi/m2-80 mCi/m2. External scintigraphy was used to evaluate the MAb biodistribution, and quantitative external scintigraphic methods were used to determine the organ and tumor radiation doses.The biodistribution studies showed enhanced 188Re-MN-14 uptake in the liver, spleen and kidneys, compared to that of 131I-MN-14. The biological T(1/2) values for 188Re-MN-14 in the blood and whole body (in hours) were 8.2 +/- 4.1 (n = 7) and 107.8 +/- 104.2 (n = 9), respectively (mean +/- s.d.). The radiation absorbed doses (cGy/mCi) delivered to the total body, red marrow, lungs, liver, spleen and kidneys were 0.5 +/- 0.05, 3.6 +/- 1.6, 2.0 +/- 0.8, 5.9 +/- 2.5, 7.1 +/- 1.9 and 8.5 +/- 2.8, respectively. Red marrow suppression was the only DLT observed. The maximum tolerated dose of fractionated doses of 188Re-MN-14 was estimated to be 60 mCi/m2.Despite its relatively increased renal and hepatic uptake, red marrow suppression is the only DLT of 188Re-MN-14. The feasibility of administering relatively high doses of 188Re on a completely outpatient basis may make this agent a preferred candidate for radioimmunotherapy.

Several investigators have reported a considerable variability in the observed hematologic toxicity after radioimmunotherapy (RAIT) with monoclonal antibodies (MoAb) given at similar amounts of radioactivity based on body surface area and/or similar radiation absorbed doses given to the red marrow. The authors investigated various factors potentially affecting hematologic toxicity after RAIT with 131I-labeled anti-carcinoembryonic antigen (CEA) MoAb to identify the statistically significant factors from those commonly perceived clinically to substantially contribute to this toxicity.Ninety-nine patients who received 131I-labeled anti-CEA MoAb for the treatment of CEA-producing cancers were assessed for platelet and white blood cell toxicity based on the common Radiation Therapy Oncology Group criteria. Multivariate regression analysis was used to identify the statistically significant factors affecting toxicity among the following variables: red marrow dose, baseline platelet and white blood cell counts, bone and/or marrow metastases, prior chemotherapy or radiotherapy, timing of prior chemotherapy or radiotherapy in relationship to RAIT, type and number of prior chemotherapeutic regimens, age, sex, antibody form, and cancer type.Red marrow dose, baseline platelet or white blood cell counts, multiple bone and/or marrow metastases, and chemotherapy 3-6 months before RAIT were the only four significant factors affecting hematologic toxicity according to multivariate analysis. The identification of bone and/or marrow metastases and recent chemotherapy as significant factors for hematologic toxicity could be important in the design of future clinical trials.

Elevated renal uptake and extended retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. However, cationic amino acids have been shown to reduce renal accretion. The aims of the current study were to evaluate whether this methodology would benefit therapy with yttrium 90 (90Y)-labeled antibody fragments (Fab, F(ab)2), to establish the relationship between radiation dosimetry and observed biologic effects, and to compare the antitumor efficacy of antibody fragments with that of whole immunoglobulin (Ig)G.The maximum tolerated dose (MTD) and the dose-limiting organ toxicity of 90Y-labeled anti-carcinoembryonic antigen (CEA) MN-14 monoclonal antibodies (Fab, F(ab)2, and IgG) were determined in nude mice bearing GW-39 human colon carcinoma xenografts. The mice were treated with or without kidney protection by administration of D-lysine, with or without bone marrow transplantation (BMT), or with combinations of each. Toxicity and tumor growth were monitored at weekly intervals after radioimmunotherapy. Dosimetry was calculated from biodistribution studies using 88Y-labeled antibody. Three different dosimetric models were examined: 1) taking solely self-to-self doses into account, using S factors for 90Y in spheroids from 0.1 to 1 g; 2) correcting for cross-organ radiation; and 3) using actual mouse anatomy as represented by nuclear magnetic resonance imaging with a three-dimensional internal dosimetry package (3D-ID).The kidney was the first dose-limiting organ with the use of Fab fragments. Acute radiation nephritis occurred at injected activitiesor = 325 microCi, and chronic nephrosis at dosesor = 250 microCi. Activities of 200 microCi were tolerated by 100% of the animals (i.e., the MTD). Application of lysine decreased the renal dose by approximately fivefold, facilitating a 25% increase in the MTD (to 250 microCi), because myelotoxicity became dose-limiting despite red marrow doses of less than 5 gray (Gy). By using BMT and lysine, the MTD could be doubled from 200 to 400 microCi, where no biochemical or histologic evidence of renal damage was observed (kidney dose,or = 40 Gy). With injected activities ofor = 325 microCi without kidney protection, and with a hepatic self-to-self dose of only 4 Gy, rising liver enzymes were observed, which could be explained only by cross-organ radiation from radioactivity in the kidneys (in the immediate neighborhood of the right kidney up toor = 150 Gy). The MTD of F(ab)2 fragments could be elevated only by a combination of BMT and lysine. With IgG, the bone marrow alone was dose-limiting. Tumor dosimetry correlated well with antitumor effects; Fab was more effective than F(ab)2, which was consistent with its more favorable dosimetry, and it may also be more effective than IgG due to its higher dose rate and more homogenous distribution. Dosimetry Model 1 was insufficient for predicting biologic effects. Model 2 seemed to be more accurate, accounting for interorgan crossfire. However, Model 3 showed an additional substantial contribution to the red bone marrow dose due to crossfire from the abdominal organs.These data show that radiation nephrotoxicity is an important effect of cancer therapy with radiometal-conjugated antibody fragments or peptides. However, this effect can be overcome successfully with the application of cationic amino acids, which substantially increase the anti-tumor efficacy of radiometal-labeled immunoconjugates. For understanding the biologic effects (e.g., liver toxicity) of 90Y in a mouse model, accounting for cross-organ radiation is essential. Further studies with radiometal-conjugated monoclonal antibody fragments and peptides are necessary to determine the MTD, dose-limiting organs, antitumor effectiveness, and nephroprotective effects of cationic amino acids in humans.

We have attempted to improve the therapeutic index of radioimmunotherapy by manipulating the blood clearance rate and the catabolism of the radiolabel. The general strategy is to allow the antibody (Ab) to circulate in the blood for 2-3 days, then to clear it rapidly by a method that delivers the Ab to hepatocytes. In addition, the radiolabel selected has two key properties: it is a residualizing label (which is lysosomally trapped after catabolism), so it is retained well by tumor cells, but is excreted rapidly by hepatocytes into bile.In initial experiments, three residualizing radiolabels were tested for their rate of excretion after specific delivery in vivo to either hepatocytes, via galactosylated Ab, or Kupffer cells, via immune complexes. A label showing rapid biliary excretion only after delivery to hepatocytes, (111)In-benzyl-diethylenetriamine tetraacetic acid, was then used for radioimmunodetection in a protocol of delayed rapid blood clearance in which clearance was by hepatocytes. This was achieved by using galactosylated Ab, combined with temporary inhibition of the asialo-glycoprotein receptor on hepatocytes. Ab RS11 and the lung adenocarcinoma Calu-3 xenograft in nude mice were used. Control experiments were performed with a conventional 125I label and with 125I-dilactitol-tyramine.Indium-benzyl-diethylenetriamine tetraacetic acid was identified as a label that was excreted more rapidly from hepatocytes than from Kupffer cells, by biliary excretion. Using this radiolabel with delayed rapid blood clearance, very high tumor/blood ratios were obtained, 166:1 at day 3, but tumor/normal tissue ratios for other tissues were not as high. There appeared to be some uptake of the radiolabel by all normal tissues tested, including the lungs and muscle. Dosimetry calculations suggested that the therapeutic index was no better than with a simple Ab injection.Antibody catabolism can be directed towards either hepatocytes or Kupffer cells, and this difference can strongly affect the excretion rate of radiolabels, since only hepatocytes can excrete degradation products into bile. Processing will also depend on the particular radiolabel. These factors are particularly important for protocols involving delayed rapid blood clearance, since liver uptake is so rapid. The methods described should stimulate other approaches of manipulating Ab blood clearance and radiolabel catabolism to achieve improved therapeutic results.

In order to optimize radioimmunotherapy (RAIT) as a cancer-treatment modality, it is necessary to select the appropriate radionuclide and antibody carrier. We evaluated the therapeutic potential of a single cycle of Mu-9 anti-CSAp monoclonal antibody (MAb) labeled with 3 different radionuclides, 131I, 90Y and 188Re. Intact antibodies and bivalent fragments with different blood clearance kinetics, normal organ distribution and varying tumor accretion and retention are also evaluated. Efficacy of treatment for large and small tumor burden was assessed in nude mice bearing s.c. GW-39 human colonic-carcinoma xenografts or intrapulmonary micrometastatic GW-39 colonies at the maximal tolerated dose of each agent. The magnitude and duration of myelosuppression associated with each radioantibody was considered by monitoring peripheral blood counts, marrow colony-forming unit activity and hematopoietic tissue weight. Radiation-dose estimates were calculated based on the kinetics of antibody accretion and elimination from tumor and normal tissues, and the results were correlated with tumoricidal activity and dose-limiting toxicity results. These studies, therefore, represent a detailed analysis, in a well-defined experimental tumor system, of several parameters (antibody form, radioisotope, tumor size) influencing the overall outcome of RAIT using equitoxic doses. It was found that myelosuppression is the primary dose-limiting toxicity for all radioantibodies except 90Y-F(ab')2, even though the different agents showed varied organ distribution. In a single-cycle treatment schedule of Mu-9 MAb, the 131I-labeled IgG is the radioimmunoconjugate of choice for the treatment of s.c. and intrapulmonary growth of the GW-39 human colonic-carcinoma xenograft in nude mice.

The goal of our studies was to determine whether administration of IL-1/GM-CSF to mice could reduce radio-antibody-induced myelosuppression and allow either dose escalation of radio-antibody using 131I, 90Y or 188Re conjugated to either intact antibody or bivalent fragments or more frequent dosing with 131I-IgG. Survival, peripheral blood counts, hematopoietic tissue weight and number of marrow CFCs were used to determine the ability to dose-intensify with a single dose or to reduce the spacing between doses. In this report, we show that in the absence of cytokines, 2 cycles of 131I-IgG spaced at 28, 35, 42 and 49 days resulted in 100%, 100%, 40% and 0% lethality, respectively. In contrast, cytokine intervention reduced lethality to 45%, 20%, 0% and 0% at the same time intervals between doses. Thus, the use of cytokines permits at least a 1 week earlier redosing of 131I-IgG. Cytokine intervention also has reduced the magnitude of myelosuppression, as measured by neutropenia and thrombocytopenia, thus permitting intensification of single doses of radio-iodinated intact antibodies, bivalent fragments and 90Y-IgG by at least 30%, 50% and 25%, respectively. However, cytokines were not effective at permitting dose escalation of either 90Y-F(ab')2 or 188Re-IgG. Further optimization of the dose schedule of cytokine administration needs to be explored for these 2 nuclide-antibody forms.

The aim of this study was to determine, in a Phase I/II clinical trial, the pharmacokinetics, dosimetry and toxicity, as well as antitumor activity, of the 131I-labeled murine anti-carcinoembryonic antigen (CEA) monoclonal antibody, NP-4 (IgG1 subtype).A total of 57 patients with CEA-expressing tumors (29 colorectal, 9 lung, 7 pancreas, 6 breast and 4 medullary thyroid cancer patients), mostly in very advanced stages, were treated. The patients underwent a diagnostic study (1-3 mg of IgG and 8-30 mCi of 131I) to assess tumor targeting and to estimate dosimetry, followed by the therapeutic dose (4-23 mg and 44-268 mCi), based on the radiation dose to the red marrow. Imaging was performed from 4-240 hr postinjection (planar and SPECT). Blood and whole-body clearance were determined; radiation doses were calculated by the Medical Internal Radiation Dose scheme.Red marrow doses ranged from 45 to 706 cGy, and whole-body doses ranged from 31 to 344 cGy. Differences in pharmacokinetics were found between different types of CEA-producing tumors: blood T 1/2 was significantly lower in colorectal cancer when compared to all other tumor types (21.4 +/- 11.1 hr versus 35.8 +/- 13.2 hr, p0.01), as was also whole-body t 1/2. Myelotoxicity was dose-limiting, and its severity was related to the types of prior therapy and extent of bone marrow involvement. In patients without prior radiation or chemotherapy, marrow doses as high as 600 cGy were tolerated without evidence of dose-limiting toxicity. No major toxicity to other organs was observed. Tumor doses were inversely related to the tumor mass and ranged between 2 and 218 cGy/mCi. Modest antitumor effects were seen in 12 of 35 assessable patients (1 partial remission, 4 minor/mixed responses and 7 with stabilization of previously rapidly progressing disease).These results suggest that prior chemotherapy or external beam radiation is an important risk factor for the development of hematological toxicity in radioimmunotherapy and that higher radiation doses may be delivered to tumors of patients without prior therapy compromising the bone marrow reserve. The different and, in the individual cases, unpredictable clearance rates suggest the necessity of dosimetry-based treatment planning rather than mCi/m2 dosing. Small tumors seem to be more suitable for radioimmunotherapy because of their favorable dosimetry, but to achieve better therapeutic results in patients with bulky disease, the application of higher, potentially myeloablative doses is indicated.

In this study, we examined the factors that may influence tumor dosimetry in the radioimmunotherapy of solid, CEA-expressing cancers.Data from 119 tumors in 93 patients with CEA-expressing cancers were analyzed. The patients underwent radioimmunotherapy with the 131I-labeled IgG1 anti-CEA antibodies NP-4 (Ka = 10(8) M-1) or MN-14 (Ka = 10(9) M-1), its humanized form hMN-14, as well as the anticolon-specific antigen-p (CSAp) antibody, Mu-9. For dosimetry, the biodistribution, targeting kinetics and cumulated activity of tumors and organs were determined from planar and SPECT imaging.An inverse logarithmic relationship between tumor size and antibody uptake was found for both anti-CEA antibodies, whereas no such relationship was found for Mu-9. The absolute tumor uptake was identified as the most important factor determining the radiation dose to the tumor (r = 0.9), with the biological half-life of the antibody in the tumor being of secondary importance (r = 0.5). No significant difference in tumor uptake was found between both anti-CEA antibodies, despite their tenfold difference in affinity. At comparable masses, colorectal and medullary thyroid cancers had significantly higher tumor uptakes (p = 0.02), as well as tumor-to-red marrow dose ratios, than other cancer types. The tumor half-lives of the anti-CEA antibodies were significantly lower in colorectal than in all other tumor types (p = 0.01).In radioimmunotherapy, tumor uptake appears to be the most important dose-determining factor. Differences in antibody affinity are reflected by differences in the biological half-life, not the absolute uptake. Especially favorable conditions for anti-CEA antibodies seem to prevail in colorectal cancer patients having minimal disease, as well as in medullary thyroid cancer, where cytotoxic tumor doses might be expected. Antimucin antibodies may have a particular advantage in the treatment of patients with larger colorectal tumors.

Attachment of 131I to MAbs through adducts such as dilactitol-tyramine (DLT), which remain lysosomally trapped after catabolism of the labeled MAb, can greatly increase the residence time of the radiolabel at the tumor site. Our previous studies demonstrated a marked increase in accretion of 131I in a human lung-cancer xenograft model, using 131I-DLT in comparison to 131I linked to MAb by the conventional chloramine-T methodology.In this study, biodistribution experiments were performed to evaluate the effect of protein dose on the accretion of 131I-DLT-labeled MAb RS11 in tumor and nontumor tissues, and in vivo radioimmunotherapy experiments compared the effect of single injections of 131I-DLT-labeled MAb RS11 to conventional 131I-labeled RS11 and an untreated control group.Dosimetry calculations based on the biodistribution data indicate only small changes in absorbed dose-to-tumor and nontumor tissues with increasing protein dose up to 100 micrograms, with a predicted absorbed dose to tumor of from 21,000 to 25,000 cGy/mCi. A single dose of 100 microCi of 131I-DLT-RS11 was found to cause tumor regression. At 7 wk postinjection of the radiolabeled MAbs, tumor volume in 73% of the animals administered 131I-DLT-labeled RS11 remained smaller than at the time of MAb injection. This is compared to 14% of the tumors in the conventionally labeled 131I-RS11 group and none in the untreated group.The use of the residualizing radiolabel DLT provides a therapeutic advantage in comparison to conventional 131I-labeled RS11.

LL1, a monoclonal antibody (MAb) to HLA Class-II-like antigen (li determinant) on the surface of B-lymphocytes, monocytes and histiocytes, was evaluated as an agent for bone marrow imaging. Six patients with diverse diseases (non-Hodgkin's lymphoma, n = 2; multiple myeloma, n = 1; polycythaemia vera, n = 1; lung cancer, n = 1; breast cancer, n = 1) were given low protein doses (1 mg) of 99Tcm (30 mCi) labelled Fab' of LL1. 99Tcm-sulphur colloid (SC) imaging was performed in three patients for comparison. Both planar and single photon emission tomographic images were acquired using Sopha gamma cameras. As early as 2 h post-MAb injection, excellent bone marrow images were achieved in all patients, demonstrating both normal or hyperproliferative marrow, as well as 'cold' bone marrow abnormalities such as radiation defects or cancer metastases. Similar to SC, relatively high uptake of LL1 was found in the liver and spleen. However, the bone marrow-to-liver and -spleen uptake ratios were approximately 19-fold higher (0.75 vs 0.04) and 6-fold higher (1.23 vs 0.22), respectively, with LL1 than with SC. The higher bone marrow uptake allowed clearly superior visualization of the thoracic spine when compared to SC. The mean T1/2 of blood and whole-body clearance were 0.4 and 66 h, respectively. The highest radiation absorbed doses (in cGy mCi-1) were observed in the spleen (0.47 +/- 0.24), kidneys (0.25 +/- 0.09) and liver (0.14 +/- 0.04). The bone marrow dose was only 0.05 +/- 0.02 cGy mCi-1. These results indicate that bone marrow imaging with 99Tcm-LL1 is feasible, and that LL1 may be a suitable alternative to SC because of better visualization of the lower thoracic spine. Potential applications include the improved detection of bone marrow metastases of solid tumours and the assessment of haematological disorders.

The hemoregulatory peptide, pGlu-Glu-Asp-Cys-Lys (pEEDCK or HP5b), has been shown to reversibly inhibit the proliferation of bone-marrow progenitor cells, and has been reported to protect mice from the myelotoxicity associated with ara-C, a chemotherapeutic agent. We undertook to use this reagent to reduce radioimmunotherapy(RAIT)-associated bone-marrow toxicity by suppressing hematopoiesis during the critical period when bone marrow is exposed to radiation. The reported studies optimize the use of HP5b to reduce the duration of neutropenia and thrombocytopenia. We found that 3.6 micrograms/day of HP5b administered through a continuous 7d mini-osmotic pump, together with a bolus dose of 3.6 micrograms 3 hours before the radioantibody dose, gave the best effect, as measured by neutrophil counts on day 28 post RAIT. With sub-lethal doses of RAIT, the period of neutropenia was reduced by 2 weeks, and there appeared to be more rapid recovery of morphologically mature myeloid cells. The peptide, however, does not appear to alleviate the lymphotoxicity associated with RAIT. No adverse effects have been noted from continuous infusions of the peptide. In the past, we reported that cytokines (IL-I/GM-CSF) are not marrow-restorative when given after RAIT. However, an additive effect is observed when HP5b infusions are combined with post-RAIT cytokine administration, suggesting that a significant pool of bone-marrow progenitor cells remains when HP5b is co-administered with RAIT. Thus, HP5b is an alternate approach to reducing myelotoxicity, and may be used in combination with cytokines to further reduce the duration of myelosuppression.

Multiple imaging methods are currently used to detect the presence, location, and extent of breast cancer, either at initial presentation or during follow-up. Even at initial diagnosis, more specific methods are needed to complement the high sensitivity of mammography in the detection and diagnosis of mammary carcinoma. Therefore, the current study evaluates the potential use of a new monoclonal antibody-based imaging agent, arcitumomab (CEA-Scan), which comprises an anti-carcinoembryonic antigen monoclonal antibody Fab' fragment labeled directly with technetium-99m, in the detection of primary, recurrent, or metastatic mammary cancer. A prospective, non-randomized, open-label, multicenter trial was conducted in 123 women 100 with primary breast cancer confirmed by histology or cytology, and 23 with a prior history and who were under evaluation for possible recurrence or metastasis. A 1-mg dose of arcitumomab labeled with 20-25 mCi Tc-99m was injected i.v., and planar images were acquired at 5-8 hr, as well as single-photon emission computed tomography. Additional planar scanning was performed optionally at 18-24 hr. The imaging studies were interpreted as positive if there was abnormal focal activity, and were correlated, wherever possible, with histopathology or conventional imaging tests. In 78 evaluable patients with primary breast carcinoma arcitumomab showed an imaging sensitivity and specificity of 82% and 88%, respectively, even detecting lesions1 cm in 61% of the cases. Among 58 patients evaluable for axillary node involvement, 30 were positive by histopathology, of which arcitumomab was correct in 19 with cancer (63% sensitivity) and in 25 of 28 without cancer (89% specificity). Among 88 primary operable patients, arcitumomab showed additional sites of focal uptake outside of the breasts and axillae in 10, or 11%. In a total of 19 breast cancer patients with known or suspected recurrence or metastatic disease, 13, or 68%, showed one or more focal sites of uptake, involving the typical sites of spread of mammary cancer. No significant adverse effects or induction of human antimouse antibodies were observed in these patients. Arcitumomab immunoscintigraphy is a simple, same-day, and safe new imaging modality for the detection of sites of breast cancer, including lesions1 cm, with a reasonably high rate of sensitivity and specificity. It may be used to complement mammography in the differentiation of malignant from benign disease.

A number of radioimmunotherapy (RAIT) trials with iodinated antibodies have shown a high variability in the radiation doses to the thyroid. Therefore, the aim of this study was to evaluate which factors influence these thyroid doses during RAIT with 131 iodinated monoclonal anti-carcinoembryonic antigen (CEA) antibodies. Data from 36 patients with CEA-expressing tumours were analysed. The patients underwent RAIT with the 131 I-labelled IgG 1 anti-CEA antibody, MN-14 (K a = 10 9 l mol -1 ) or its F(ab') 2 fragment (activity range 45.8-220.0 mCi). The thyroid was blocked with 120 mg iodine (Lugol's or SSKI solution) and 400 mg perchlorate per day, starting 1 day prior to the first study. Blood clearance and molecular composition of labelled plasma compounds were determined by blood sampling and size-exclusion high-performance liquid chromatography analysis. The cumulated activities of tissues were determined from daily imaging and blood clearance data. Doses were derived from the MIRD scheme. Thyroid radiation doses showed a high variability, between 1.2 and 37.7 cGy mCi -1 (mean ± S.D. : 11.1 ± 8.3 cGy mCi -1 ), corresponding to absolute doses between 2.5 and 43.6 Gy. However, the maximal iodine uptake in the thyroid was 2.4 ± 1.9 μCi mCi -1 (range 0.2-10.0 μCi mCi -1 ), which was less than 1% of the injected activity, indicating that more than 99% of the thyroid was blocked in all cases. No correlation was found between these thyroid doses and conditions leading to an enhanced exposure to free radioiodine, such as unbound I - in the mAb preparation, rapid metabolic breakdown of the labelled antibody due to human anti-mouse antibodies (HAMA), or immune complex formation with circulating antigen. However, a relationship between the thyroid doses and the patients' compliance in taking their Lugol's and perchlorate blocking medications, as well as to a relatively high variability in the biological half-life of the iodine in the thyroid (range from 31.1 h to virtual infinity), is indicated. No rising TSH titres or other signs of (latent) hypothyroidism were seen in these patients during a 2 year follow-up period. Longer follow-up was not possible because of the terminal condition of most of the patients. These data show that thyroid doses in an appropriately blocked individual given a standard, non-myeloablative dose of RAIT, are generally lower than those assumed to be required to cause late hypothyroidism. Even if higher activities are used, potential hypothyroidism may be overcome easily by hormone replacement. Thyroid doses are independent of parameters leading to an enhanced exposure of the thyroid to free radioiodine, suggesting that patient compliance in taking their blocking medication may be the most crucial factor for reducing thyroid doses in RAIT with 131 I-labelled antibodies.

The clinical feasibility of radioimmunotherapy (RIAIT) was assessed in patients with metastatic, carcinoembryonic antigen (CEA)-producing cancers who had minimal residual or small-volume disease (tumor lesionsor = 3 cm in diameter).Thirteen cancer patients (8 colorectal, 3 lung, 1 pancreatic and 1 medullary thyroid cancer) received RAIT with 131I-NP-4 F(ab')2 anti-CEA antibody. The radioactive dose given was based on a prescribed radiation dose to the red marrow. Ten of the 13 patients received initial therapeutic doses delivering 150-450 cGy to the red marrow (70-296 mCi) and six patients had more than one therapy infusion.Targeting of all known tumor lesions0.5 cm [corrected] in diameter was possible in nine patients and at least one tumor lesion was evident in all patients. Disease stabilization ranging from 3.5 to 7 mo was seen in 6 of the 13 patients who previously had clear evidence of progressive disease. Four of the six patients with disease stabilization received the presumed maximum tolerated dose of 450 cGy to the red marrow. Red marrow suppression was the only observed toxicity and there was a good correlation between the red marrow dose and myelotoxicity. Red marrow dosesor = 250 cGy resulted inor = grade 2 myelotoxicity and a red marrow dose of 450 cGy resulted in reversible grade 3 or 4 myelotoxicity in 3 of 6 patients. Human anti-mouse antibodies (HAMA) developed in all but one of the six patients who received multiple therapeutic infusions of the antibody.RAIT of patients with small-volume disease is feasible and these patients should be considered for future dose-intensification trials because of their generally poor prognosis.

The initial clinical experience with the second-generation, high-affinity, MN-14 immunoglobulin (IgG) anticarcinoembryonic antigen (CEA) monoclonal antibody (MoAb) in patients with CEA-producing tumors was reported previously. A bivalent fragment of this MoAb, MN-14 F(ab)2, was prepared, and its pharmacokinetics, targeting properties, dosimetry, and immunogenicity were investigated.MN-14 F(ab)2(0.6-29 mg) was labeled with 131I(7.7-269 millicuries and injected into 28 patients with CEA-producing cancers. External scintigraphy was used to evaluate tumor targeting. Quantitative external scintigraphy methods were used to determine the organ and tumor radiation doses.The overall sensitivity of tumor targeting on a lesion basis was 86%, similar to that reported previously for MN-14 whole IgG. The biologic T1/2's for the fragment in the blood and total body (in hours) were 16.8 +/- 4.1 and 59.4 +/- 9.4, respectively, compared with 27.3 +/- 15.7 and 69.6 +/- 32.2 reported for MN-14 IgG. Depending on the protein dose given, high plasma CEA levels (100ng/ML) resulted in a significant alteration of MoAb pharmacokinetics and organ dosimetry. Individual tumors received an average dose of 10.7 +/- 7.3 centigray [cGy]/mCi, and the tumor-to-total body, red marrow, lung, liver, and kidney dose ratios were 16.8 +/- 11.1, 5.6 +/- 3.6, 5.1 +/- 3.9, 6.0 +/- 3.8, and 3.1 +/- 2.0, respectively (mean + standard deviation [SD]). Only 9 of 18 patients (50%) injected with4 mg (range: 4-52.1 mg) of MN-14 F(ab)2 developed significant levels of human antimouse antibodies, suggesting that the F(ab)2 may be less immunogenic than the intact IgG.MN-14 F(ab)2 exhibits a similar targeting sensitivity and tumor dose as reported previously for the IgG form. The lower red marrow doses combined with lower immunogenicity expected for this agent, may make it a suitable alternative for future imaging and therapeutic applications.

This study evaluates the pharmacokinetics, dosimetry, toxicity and therapeutic potential of radiolabeled NP-4 and MN-14 anti-CEA antibodies in medullary thyroid cancer (MTC).Eighteen patients with advanced MTC entered exploratory clinical studies with therapeutic doses of 131I-labeled NP-4 and MN-14 murine monoclonal antibodies (MAbs) reactive with carcinoembryonic antigen (CEA). Doses administered ranged from 46 mCi for 131I-MN-14 lgG to 195 mCi for 131I-MN-14 F(ab)2 in patients negative for human anti-mouse antibodies (HAMA).The radioconjugate blood half-life (T1/2) for the whole lgG was 42.5+/-5.0 hr compared to 18.8+/- 4.1 hr for the bivalent fragments. Tumor doses of 17.5+/-11.0 and 11.4+/-6.3 cGy/mCi were estimated for 131I-MN-14 lgG and F(ab)2, respectively. Tumor/red marrow dose ratios exceeded 3:1 for most lesions. Red marrow doses of up to 350 cGy generally could be delivered withgrade 4 toxicity. Seven of 14 evaluable patients showed evidence of anti-tumor effects lasting up to 26 months, based on physical exam, tumor markers or computed tomography.This study demonstrates that anti-CEA MAbs may be suitable for radioimmunotherapy of metastatic or recurrent MTC.

The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments.The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique).The renal uptake in the amino acid group was significantly lower (p0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group.The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.

The targeting potential of 131I-labeled NP-4 and MN-14 anti-CEA (carcinoembryonic antigen) monoclonal antibodies (MAbs) was assessed in 19 patients with metastatic medullary thyroid cancer (MTC). Seventeen of these patients also entered pilot radioimmunotherapy studies with nonmyeloablative doses of 131I-anti-CEA MAbs. Tumor targeting was possible in all 19 patients, with an overall lesion sensitivity of 91%. Tumor dosimetry with 131I-MN-14 IgG or F(ab)2 was very favorable, with tumor doses of 14.3 +/- 8.3 cGy/mCi and tumor:red marrow dose ratios exceeding 3:1 for most lesions. Limited antitumor effects lasting up to 26+ months, based on physical exam, tumor markers, computed tomography, or a followup MAb scan, were seen in 5 of 11 assessable patients given relatively low doses of 131I-labeled anti-CEA MAbs. We conclude that anti-CEA MAbs are excellent agents for targeting metastatic MTC. The high tumor uptake of the 131I-anti-CEA antibodies and evidence of tumor response in some patients suggest that radioimmunotherapy with radioiodinated anti-CEA MAbs may be an effective treatment for MTC, particularly when the maximum tolerated dose is given alone or in combination with autologous red marrow or peripheral stem cell support.

The value of 67Ga citrate scanning as a transferrin receptor agent was compared in this study with a 99mTc-labeled anti-CD22 (B-cell) Fab' fragment (LL2) in patients with low- and high-grade B-cell non-Hodgkin's lymphoma (NHL). Thirteen patients with histologically confirmed NHL were examined prospectively with both radiopharmaceuticals within 8 days. The results of immunoscintigraphy were compared with those of 67Ga scanning and the clinical and radiological workup (computed tomography, ultrasound, and magnetic resonance imaging) of the patients. The overall sensitivity of 67Ga citrate and 99mTc-labeled LL2 fragment was each 80% in a total of 43 lesions. Low-grade lymphoma patients had a higher sensitivity in LL2 imaging (82% versus 71%), and high-grade lymphoma patients in 67Ga citrate scanning (100% versus 75%). The target:background ratio in low-grade NHL for LL2 was 1.43 +/- 0.3:1 versus 1.8 +/- 0.5:1 in 67Ga scans; in high-grade NHL, 1.49 +/- 0.35:1 versus 2.2 +/- 0.8:1, respectively. Single-photon emission computed tomography imaging was necessary in 21.7% of the patients 4 h after injection to localize the lesions. In conclusion, the overall sensitivity of 99mTc-labeled LL2 is comparable to 67Ga citrate scanning in patients with B-cell NHL. 99mTc-labeled LL2 antibodies are rapid to use, are safe, and need a shorter imaging time (24 h versus 72 h). Because of these advantages, 99mTc-labeled LL2 may be superior to 67Ga scanning for the staging of lymphoma patients.

Radioimmunodetection (RAID) with the 99mTc-labeled Fab' fragment of monoclonal antibody (MoAb) LL2 has been reported to have a high lesion detection rate for malignant lymph nodes as well as for visceral and skeletal tumor masses (20). Our purpose in this study was to evaluate the safety and staging efficacy of the 99mTc-labeled Fab' fragment of MoAb LL2 in patients with various grades and stages of B-cell non-Hodgkin's lymphoma (NHL). Thirty adult patients, 13 male and 17 female, ranging in age from 20 to 80 years, with at least one biopsy-proved malignant node (or = 0.5 cm) and a Karnofsky performance score ofor = 60% were enrolled in this study. Patients underwent selected planar and single photon emission computed tomographic imaging at 6 and 18 h after receiving an i.v. infusion of 0.25-1 mg of LL2 Fab' fragment labeled with 25-30 mCi of 99mTc. RAID findings were compared with physical examination, chest radiography, computed tomography, magnetic resonance imaging, and bone and 67Ga scan findings. The RAID scan was positive in all but three patients. The sensitivity for known lesions was 90% and for suspected lesions, 89%, with an overall positive predictive value of 96%. Twenty-nine of the 30 patients had either low- or intermediate-grade NHL. Fifteen of 16 (94%) low-grade patients were correctly staged by RAID; three of these patients were correctly upstaged. Twelve of the 13 (92%) intermediate-grade patients were correctly staged by RAID; two of these patients were correctly upstaged. The high-grade NHL patient was staged correctly by RAID. Infused doses of 99mTc-labeled LL2 Fab' of 0.5 and 1.0 mg did not affect lesion sensitivity. The RAID sensitivity decreased as the total tumor burden increasedor = 100 g. On the basis of these initial results, the 99mTc-labeled Fab' fragment of MoAb LL2 (LymphoScan) seems to yield useful clinical information, especially for the staging of B-cell NHL patients who do not have bulky disease (i.e., tumor burdens ofor = 100 g).

The renal uptake of radiolabeled antibody fragments and peptides is a problem in radioimmunodetection and radioimmunotherapy, especially with intracellular retained radiometals. The aim of this study was to develop suitable methods to reduce this kidney uptake. BALB/c mice or nude mice bearing the human GW-39 colon carcinoma xenograft were given i.p. injections of basic amino acids or a range of different basic amino acid derivatives, amino sugars, as well as cationic peptides. The effect of these agents on the biodistribution of Fab' and F(ab')2 fragments of different mAbs radiolabeled with 99mTc, 188Re, 111In, 88Y, or 125I was studied. Tumor and organ uptake was determined and compared to untreated mice. The kidney uptake of Fab' fragments was reduced 5-6-fold in a dose-dependent manner as compared to untreated controls. The uptake in all other organs, as well as the tumor, was unaffected. A similar reduction in renal retention was seen for all other intracellularly retained isotopes, as well as for F(ab')2 fragments. D- and L-isomers of lysine were equally effective whether given i.p. or p.o. D-glucosamine was effective, but its N-acetyl derivative was not. Basic polypeptides (e.g., poly-L-lysine) were also effective; their potency increased with increasing molecular weight. HPLC of the urine taken from treated animals showed the excretion of intact Fab', in contrast to mostly low-molecular-weight metabolites in the control group. These studies indicate that a variety of basic compounds is capable of inhibiting the tubular reabsorption of peptides and proteins, thus lowering the kidney uptake of antibody fragments significantly. On a molecular basis, the effect seems to essentially rely on the presence of a positively charged amino group. By reducing renal retention of antibody fragments, their role as imaging and therapeutic agents may be expanded.

The effect of using a "residualizing" iodine radiolabel, dilactitol-iodotyramine, for radioimmunolocalization of antibodies to tumors was investigated. This tracer is designed to be lysosomally trapped after catabolism of the labeled antibody. mAbs RS7 and RS11 were used for in vivo and in vitro studies on the uptake and retention of radioisotope into tumor cells. Both are murine IgG1 mAbs with pancarcinoma reactivity, which react with integral membrane glycoproteins. mAb RS7 has been shown to be relatively rapidly catabolized by the antigen-bearing cell line Calu-3, whereas RS11 is catabolized more slowly in the same cells. An 111In- or 88Y-p-isothiocyanatobenzyl-diethylenetriamine pentaacetic acid conjugate was also tested because these radiometals are known to be lysosomally trapped, and iodination via chloramine T was used to provide a baseline. In vitro, a substantial increase in retention of the label by cells was observed when the dilactitol-tyramine DLT- or 111In-labeled mAbs were used, and the improvement gained by the use of these residualizing labels was greater with the use of the rapidly catabolized mAb (RS7) than it was with the more slowly catabolized mAb (RS11). In biodistribution studies in nude mice bearing Calu-3 tumor xenografts, a dramatic improvement in the tumor accretion of the radiolabel was seen with the use of the 131I-labeled DLT- or 88Y-labeled mAbs. For example, at day 7 the percentage of injected dose/g in the tumor was 5.54 +/- 1.47% (SD), 38.06 +/- 8.04%, and 43.18 +/- 19.50% for the conventionally iodinated, DLT- and 88Y-labeled RS7, respectively. Dosimetry calculations performed on the biodistribution data predict increases of approximately 8- and 4-fold in the absorbed dose to tumor with the use of 131I-labeled DLT- and 90Y-labeled mAbs, respectively, compared to the conventional 131I. In contrast to in vitro findings, these results were similar for both RS7 and RS11, suggesting that the use of DLT may be advantageous for most of the mAbs binding to the cell surface, including antibodies that are catabolized relatively slowly. The advantage of 131I-labeled DLT over 90Y is due to the longer physical half-life of the 131I.

Imaging of osteomyelitis and soft-tissue infections can be problematic with currently available agents; bone scans are often false-positive. Indium-111-oxine and 99mTc-HMPAO white blood cell (WBC) scans require ex vivo handling of blood with potential exposure to infectious agents, and 99mTc-antigranulocyte (IgG1) antibodies need 24 hr for final diagnosis.We investigated the use of 99mTc-murine anti-granulocyte monoclonal Fab' fragment in 20 patients with suspected osteomyelitis of soft-tissue infections. All patients also had 99mTc bone scans and 111In-oxine or 99mTc-HMPAO white blood cell scans. The final diagnosis was confirmed by culture, biopsy, surgery, follow-up, x-rays, CT or MRI. In vitro studies performed on granulocytes demonstrated no effect on their function when the anti-granulocyte monoclonal antibody fragment was added.Sensitivity, specificity and diagnostic accuracy to detect infection was 88%, 75% and 80%, respectively. All lesions could be detected as early as 1 hr after injection of the antibody fragment. In comparison, WBC scanning had values of 86%, 78% and 81%, respectively. Some lesions could only be detected 24 hr following the injection of labeled WBCs. LeukoScan had three false-positives and WBC scanning had two false-positives.Immunoscintigraphy with 99mTc-NCA-90 Fab' fragments offers rapid localization of foci, rapid and simple use, a negligible HAMA response rate, no effect on granulocyte function and an accuracy comparable to WBC scanning.

The diagnosis of Pneumocystis carinii pneumonia (PCP) currently relies upon cytological demonstration of the organism in sputum or bronchoscopy specimens. The purpose of this study was to develop a radiolabeled monoclonal antibody (Mab) against Pneumocystis carinii (P. carinii) and to evaluate its use for imaging PCP.We studied 16 HIV-infected patients with pneumonia in order to evaluate a new Mab-based imaging method for diagnosing PCP. Most patients were managed for opportunistic pneumonia associated with AIDS, including standard cytological tests, and, in all cases, intensive chemotherapy. Prior to the clinical study, the Mab raised to P. carinii was shown to react with human P. carinii but not with rat P. carinii or human white blood cells.After labeling a 1-mg Mab Fab' fragment with 30 mCi of 99mTc, the presence or absence of PCP could be confirmed in six of seven or seven of eight assessable patients, respectively, by external photoscanning within 24 hr. This shows a sensitivity of 85.7% and a specificity of 86.7%.Our findings suggest that PCP can be diagnosed by a noninvasive imaging method employing a small dose of a 99mTc-labeled Mab showing specificity for the infectious organism, since patients with P. carinii-free pneumonia were correctly negative in 87.5% of cases. Rapid diagnosis and organ-localization of other infectious lesions with organism-specific, radiolabeled Mabs may be feasible.

Processing radiolabeled degradation products is the key factor affecting retention of antibodies within the cell. In this study, we have analyzed the processing of antibodies labeled in nine different ways.Antibodies were labeled with three different radioisotopes and seven different forms of 125I. Eight of the radiolabels (except 188Re) were conjugated to the same antibody, MA103, and tested on the renal carcinoma cell line SK-RC-18 and/or the ovarian carcinoma cell line SK-OV-6. Rhenium conjugation utilized the antibody RS7, the target cell line ME180 and three of the other radiolabels were also tested with this antibody-target cell combination for comparison.Iodine conjugated to antibodies by conventional methods was rapidly released from the cell after antibody catabolism. In contrast, iodinated moieties, such as dilactitol-tyramine and inulin-tyramine were retained within cells four to five times longer.The use of radiolabels that are trapped within cells after antibody catabolism can potentially increase the dose of radiation delivered to the tumor, from the same amount of radioactivity deposited by a factor of four or five. The prolonged retention of 111In relative to 125I is not due to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes.

Colon-specific antigen-p is a tumor-associated antigen present in approximately 60% of colorectal cancers. Preclinical studies have shown that the murine monoclonal antibody Mu-9 has excellent tumor-targeting abilities; therefore, clinical studies were initiated.The immunoglobulin G and F(ab')2 were radiolabeled with 131I and administered to 13 and 12 patients, respectively, with advanced gastrointestinal cancer (colon, rectal, and pancreatic) for radioimmunodetection or radioimmunotherapy.Even in patients with highly elevated carcinoembryonic antigen levels, only one patient showed appreciable complexation of the labeled antibody, suggesting the epitope may not be highly expressed in the blood. Fifty percent of 131I-Mu-9 immunoglobulin G was cleared from the blood within 41 +/- 13 hours, while it took only 19 +/- 8 hours for the same amount of 131I-F(ab')2 to be cleared from the blood. Lesion detection in the abdomen, liver, and pelvis was greater than 90% for either the immunoglobulin G or F(ab')2. The dose absorbed by the normal organs, except the kidneys, was two- to threefold less for the F(ab')2 than for the whole immunoglobulin G. The dose to the kidneys was similar for both forms of immunoglobulin. The average tumor dose for 131I-Mu-9 immunoglobulin G was 13.9 +/- 11.0 cGy/mCi, and for 131I-F(ab')2 was 4.9 +/- 2.9. Tumor/red marrow dose ratios for the whole immunoglobulin G were 4.3 +/- 3.0, compared to 3.3 +/- 1.9 for the F(ab')2, suggesting the therapeutic window for the two forms of immunoglobulin may be similar. Eight of nine patients given the whole immunoglobulin G developed highly elevated levels of human anti-mouse antibody, whereas lower values were observed in five of seven patients given the F(ab')2.These initial results support the need for further evaluation of Mu-9 immunoglobulin G and F(ab')2 for targeting gastrointestinal cancer for radioimmunotherapy.

Antibody sulfhydryl groups can act as effective carriers of reduced technetium and rhenium species for radioimmunodetection and radioimmunotherapy.Intact immunoglobulin G and fragments were labeled with the isotopes and examined in vitro and in vivo.Technetium bound to intact immunoglobulin G was found to be the most stable species in vitro, but in vivo, clearances of technetium and rhenium bound to intact antibody were similar. Serum clearances were faster than those seen for the corresponding radioiodinated antibodies. In vivo clearance rates of the radiolabeled fragments were similar, with kidney uptake and retention seen. Rhenium-labeled antibodies, despite a greater tendency toward in vitro reoxidation than technetium-labeled antibodies, did not show enhanced kidney clearance in animal models. Rhenium-188 and technetium-99m were obtained from similar generator systems in carrier-free form. Using rhenium-188 spiked with cold rhenium, it was determined that approximately one rhenium atom per molecule of antibody can be conjugated directly. Rhenium-186 also was coupled at almost a 1:1 ratio to antibody.Only radiolysis concerns will limit the amount of rhenium-188 conjugated to antibody. Large doses of antibody will be necessary to deliver rhenium-186 at this isotope's currently available specific activity. Otherwise, higher specific activity rhenium-186, and/or greater loading capacity of rhenium-186 onto antibody, will be needed to generate the type of product that will be usable at a clinical dose of several hundred millicuries.

RS7 is a murine monoclonal antibody immunoglobulin G1 with pan-carcinoma reactivity, which was raised against human squamous cell carcinoma of the lung. To optimize the use of monoclonal antibody RS7 as a carrier of radionuclides for tumor targeting and therapeutic applications, whole RS7 immunoglobulin G and its F(ab')2 fragment were radiolabeled, and their biodistribution and effectiveness as radioimmunotherapeutic agents in nude mice bearing established human tumor xenografts were evaluated. The contributions of the tumor model, monoclonal antibody form (fragment vs. intact), radioisotope (131I, 111In, 90Y, and 188Re), and antigen target were evaluated.Cumulative absorbed radiation doses were calculated from biodistribution data, and doses were normalized to blood to estimate expected relative toxicities. Two tumor models expressing different levels of RS7-antigen were studied: ME180, a cervical carcinoma cell line, and Calu-3, an adenocarcinoma of the lung cell line. In addition, the therapeutic effectiveness of 131I-RS7-F(ab')2 was compared to that of 131I-RS7-IgG.Doses delivered to tumor (normalized to blood) calculated for 131I-RS7-F(ab')2 and 90Y-RS7-IgG were 4.7 times and 1.8 times greater, respectively, than 131I-RS7-IgG, and therefore would be expected to yield greater therapeutic efficacy when equitoxic doses are administered. This expectation was confirmed in the radioimmunotherapy study with 131I-RS7-F(ab')2. At equivalent absorbed dose to tumor, 131I-RS7-F(ab')2 was found to effect a slightly longer suppression of tumor growth than the intact 131I-RS7 IgG, and a 50% dose escalation yielded tumor regression for a prolonged period with the fragment, whereas a similar 50% dose escalation with 131I-RS7-IgG could not be tolerated.

The authors recently reported that a 12-day schedule (beginning 3 days before radioantibody treatment) of twice-daily dosing of rH-IL-1 (1 x 10(3) U/dose) and rM-GM-CSF (0.5 micrograms/dose) can reduce the magnitude and duration of radioantibody-induced myelosuppression, thereby permitting a 25-30% increase in the dose of radioantibody that can be administered without the dose proving lethal. In an effort to further reduce toxicity and escalate the tolerated dose, the authors altered the method of administration of cytokines from daily bolus dosing to continuous infusion by implantable osmotic pumps.A control group of mice was compared to five groups of mice that either did or did not receive a 340 microCi dose of radioantibody, and received no cytokines, cytokines by bolus dosing, or cytokines by continuous infusion. For 4 weeks, peripheral white blood cell and thrombocyte counts and thymus and spleen weights were taken, marrow cell number was monitored, and marrow colony-forming unit activity was evaluated weekly in the untreated control mice and the treated mice.These studies demonstrated that after a dose of radioantibody, continuous dosing of cytokines resulted in higher white blood cell (WBC) and platelet values than if bolus delivery was used (day 7, WBC: 110% vs. 59%; day 14, WBC: 85% vs. 62%; day 21, WBC: 98% vs. 42%; day 7, platelets: 122% vs. 51%; day 14, platelets: 159% vs. 72%; day 21, platelets: 239% vs. 171%). A comparison of bolus versus continuous dosing in the absence of radioantibody indicated that spleen weight increased by 40-60% after continuous infusion of cytokines and by 20-25% after bolus dosing. The 20-30% decrease in thymus weight was similar with both dosing regimens. Colony-forming units (CFUs) in marrow increased from 30-35 in untreated mice to 50-55 in mice given cytokines by bolus injection, and to 150-180 in mice given continuous infusion of cytokines. Spleen CFUs exhibited an insignificant increase after bolus dosing of cytokines but increased almost fourfold after continuous dosing. Peak stimulation of marrow and spleen CFUs occurred 28 days after initiation of cytokine administration (2 weeks after cytokines administration was stopped). The probability of survival for 6 weeks after further dose escalation to 360 microCi I-131-MN-14 immunoglobulin G was 16.4% +/- 8.6% after bolus dosing and 58.1% +/- 11.3% after continuous infusion of cytokines.Although continuous infusion of cytokines proved to be a better method of reducing hematopoietic toxicity, further dose escalation of radioimmunotherapy using the "pump" method of cytokine delivery was not possible. Cytokine intervention by either mode of delivery permits a 25% dose intensification without the dose becoming lethal. Further escalation is not feasible, possibly because of other end organ toxicity.