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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.

This article reviews the development of radioimmunoconjugates as a new class of cancer therapeutics. Numerous conjugates involving different antigen targets, antibody forms, radionuclides and methods of radiochemistry have been studied in the half-century since radioactive antibodies were first used in model systems to selectively target radiation to tumors. Whereas directly conjugated antibodies, fragments and subfragments have shown promise preclinically, the same approaches have not gained success in patients except in radiosensitive hematological neoplasms, or in settings involving minimal or locoregional disease. The separation of tumor targeting from the delivery of the therapeutic radionuclide in a multistep process called pretargeting has the potential to overcome many of the limitations of conventional, or one-step, radioimmunotherapy, with initial preclinical and clinical data showing increased sensitivity, specificity and higher radiation doses delivered. Our particular focus in pretargeting is the use of bispecific, trimeric (three Fab′s) constructs made by a new antibody engineering method termed ‘dock-and-lock.’

The efficacy of radiation treatment depends upon local oxygen concentration. We postulated that the variability in responsiveness of tumor xenografts to a fixed dose of radioimmunotherapy might be related to the tumor pO2 at the time that radioimmunotherapy was administered. We evaluated the growth of xenografts of CALU-3 tumors, a non-small cell lung carcinoma, in response to an 8.9-MBq dose of 131I-RS-7-anti-EGP-1 and correlated tumor growth rate with initial tumor pO2 measured by EPR oximetry. The greatest growth delay in response to radioimmunotherapy had the highest initial pO2, and the fastest-growing tumors had the lowest initial pO2. We then determined the dynamic effect of radioimmunotherapy on tumor pO2 by serial measurements of pO2 for 35 days after radioimmunotherapy. This information could be important for ascertaining the likelihood that a tumor will respond to additional doses as part of a multiple dose scheme. Serial tumor pO2 measurements may help identify a window of opportunity when the surviving tumor regions will be responsive to a second round of radioimmunotherapy or a second therapeutic modality such as chemotherapy or an anti-vascular agent. After radioimmunotherapy, there was an increase in tumor pO2 followed by a decrease below initial levels in most mice. Thus defined times may exist when a tumor is more or less radiosensitive after radioimmunotherapy.

We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type.

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.

A natural N-linked glycosylation site (Asn-Val-Thr) at amino acid positions 18-20 (Kabat's numbering) was identified in the framework-1 (FR-1) region of the light chain variable (V kappa) domain of a murine anti-B cell lymphoma Ab, LL-2. Our earlier studies demonstrated that no contact between the V kappa-appended oligosaccharide and the Ag binding site was evident, because glycosylation at this site did not affect the Ag binding property of the Ab. By using the murine LL-2 F(ab')2 fragment (which is devoid of constant region-appended oligosaccharide) as substrate, as much as five bifunctional chelator molecules per F(ab')2 fragment could be site specifically conjugated at the V kappa-appended carbohydrate moiety with no reduction in immunoreactivity. The resulting conjugates labeled efficiently with both 90Y and 111In, with no significant effect on Ab affinity. In contrast, conjugation of less than five chelates/Ab fragment randomly at lysine residues resulted in a three- to fivefold reduction in affinity. By a single Arg to Asn mutation, an N-linked glycosylation site similar to that of LL-2 was introduced in the FR-1 segment of a nonglycosylated, humanized anti-carcinoembryonic Ag (CEA) Ab, MN-14 (hMN-14). Glycosylation at the engineered carbohydrate-addition site was demonstrated by SDS-PAGE analysis. Neither glycosylation nor site-specific conjugation of chelate at the V kappa-appended carbohydrate moiety resulted in the loss of immunoreactivity. The glycosylated hMN-14 conjugate labeled efficiently with 90Y.

Background. Various monoclonal antibodies (MoAb) labeled with Iodine-131 or Indium-111 (In-111) have been investigated for radioimmunodetection of Hodgkin's and non-Hodgkin's lymphomas. Successful radioimmunotherapy also has been reported. The purpose of this pilot study was to stage non-Hodgkin's B-cell lymphomas (NHL) using whole body scintigraphy with technetium-99m (Tc-99m)-labeled murine monoclonal antibody LL2 (EPB-2) Fab' (Immunomedics, Morris Plains, NJ). Others have shown this MoAb to have specific binding to B-cell lymphomas by flow cytometry and immunofluorescence. Initial clinical studies by others have demonstrated targeting of NHL with the Tc-99m-labeled LL2-Fab'

Trials of radiolabeled antibody therapy in patients with B-cell lymphoma have been the most promising of any in radioimmunotherapy. Response rates of greater than 90% with many complete remissions have been reported by several groups using either low (185-370 MBq) or high (8.6-22.5 GBq) doses of I-131-labeled antibodies against B-cell antigens. Estimated doses delivered to normal organs have ranged from 0.2 to 2.2 mGy/MBq and have shown similar interpatient variation in all series, despite differences in antibody specificity and dosimetric techniques. Tumor doses have ranged from 0.5 to 5.4 mGy/MBq. There has been little correlation of tumor response with estimated tumor dose. Toxicity has been limited to bone marrow suppression which has been greater with the higher amounts of I-131. An advantage for a particular antibody specificity or for high dose compared to multiple low doses has yet to be demonstrated.

Two radiolabeled antibody products for the treatment of non-Hodgkin's lymphoma have been approved, thus indicating that cancer radioimmunotherapy (RAIT) has finally come of age as a new therapeutic modality, exemplifying the collaboration of multiple disciplines, including immunology, radiochemistry, radiation medicine, medical oncology, and nuclear medicine. Clinical trials are showing usefulness in other hematological neoplasms, but the treatment of solid tumors remains the major challenge, since the doses shown to be effective in hematological tumors are insufficient in the more common epithelial cancers. Nevertheless, use of RAIT in locoregional applications and in the treatment of minimal residual disease have shown promisingThere is also optimism that pretargeting procedures, including new molecular constructs and targets, will improve the delivery of radioactivity to tumors with less hematologic toxicity, and thus may become the next generation of RAIT.

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.

There is currently no method to cure patients with disseminated colorectal cancer, which is the third leading cancer killer in the Western World. This report shows that the GW-39 intrapulmonary micrometastatic human colonic cancer model in nude mice can be cured with radiolabeled antibodies against carcinoembryonic antigen, and that this approach of radioimmunotherapy is superior to conventional chemotherapy with 5-fluorouracil and leucovorin (5-FU/LV). Monovalent Fab fragments labeled with 131I are superior to intact IgG when survival was evaluated 3, 7, and 14 days after implantation, leading to cures in up to 90% of the mice. Histological results provide support for the differences in therapeutic efficacy observed. Microautoradiography was used to evaluate the intratumoral distribution of each form of antibody. The enhanced tumor control by Fab compared with IgG could be explained in part by the homogeneity of radioantibody distribution of Fab. Biodistribution analysis and initial dose rate calculations for all three forms of antibody also help explain the ability of 131I-labeled Fab to provide better tumor growth control than seen with 131I-labeled IgG. Thus, radioimmunotherapy may be a new modality to treat metastatic disease, particularly when using small antibody fragments.

We demonstrated previously that human B-cell lymphomas were effectively and specifically killed in vitro by an antibody to CD74 (LL1) linked to (111)In or other Auger electron emitters. This study was intended to more accurately compare the potency and specificity of 3Auger electron emitters, (111)In, 67Ga, and 125I, and to evaluate beta-particle emitters, 131I and 90Y. The unique property of LL1 is its high level of intracellular uptake.Raji B-lymphoma cells were incubated with serial dilutions of the radiolabeled Abs for 2 d and then monitored for cell growth by 2 assays: a cell counting assay and a clonogenic assay. The uptake of radioactivity per cell was monitored at various time points, and the radiation dose was calculated using published S values for radioactivity located in the cytoplasm. Both specific and nonspecific toxicity were evaluated.The beta-particle emitters had considerably higher levels of nonspecific toxicity than the Auger electron emitters, but both 131I and 90Y, and particularly 131I, still had high levels of specificity. Both of these results were consistent with dosimetry calculations. Relative to the delivered disintegrations per cell, 131I and 67Ga were the most potent of the radionuclides tested, with 125I and (111)In being significantly weaker and 90Y being intermediate. The high potency of 67Ga, together with its low nonspecific toxicity, caused this radionuclide to have the highest specificity index.When delivered by Ab LL1, both Auger electron and beta-particle emitters can produce specific and effective toxicity. The choice of the optimal radionuclide for therapy may depend on the ease and efficiency of labeling, the specific activity obtained, the nature of the tumor being targeted, and other factors, but the high specificity indices of the Auger electron emitters may be an advantage.

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.

During the recovery period after anticancer myelosuppressive therapy, hematopoietic progenitor cells become mitotically active in order to replenish the bone marrow compartment and remain hyperproliferative even after normalization of peripheral white blood cells and platelets. At this stage, the progenitors are more radiosensitive and chemosensitive. Dosing patients with additional cytotoxic therapy during this phase will likely result in more severe toxicity. For example, the authors have noted that the red bone marrow (RM) dose resulting from radioantibody therapy does not correlate with observed bone marrow toxicity. Several patients given similar RM doses had Grade 3 or 4 toxicity, whereas others had Grade 0-2 toxicity even though their white blood cell (WBC) and (PLT) counts were normal at the time of dosing. The goal of these studies was to establish a noninvasive predictive marker of bone marrow activity that could determine stem cell and progenitor cell recovery from previous myelosuppressive therapy.A retrospective study was conducted to quantitate plasma levels of 5 cytokines regulating hematopoiesis, namely, 2 stimulatory fms-like tyrosine kinase (FLT3-L) and stem cell factor (SCF) and 3 inhibitory growth factors tumor necrosis factor-alpha (TNFalpha), tumor growth factor-beta, and macrophage inflammatory protein (MIP-1alpha), by immunoassay in 43 patients enrolled in clinical trials at Garden State Cancer Center in Belleville, New Jersey. All patients had had previous chemotherapy with a duration of 1-24 months. The serum cytokine values were correlated with the magnitude of leukopenia or thrombocytopenia following a single dose of radioantibody as the cytotoxic therapy.Plasma FLT3-L levels predicted excess platelet toxicity in 13 of 16 patients (mean = 225 +/- 106 pg/mL) and resulted in a false-positive in only 3 of 27 other patients (mean = 80 +/- 41 pg/mL). Plasma FLT3-L135 pg/mL resulted in 81% sensitivity and 89% and 86% specificity and accuracy, respectively, for predicting excess toxicity caused by additional cytotoxic therapy. The positive likelihood ratio was 7.5 (95% confidence interval, 2.5-22.5) and the negative likelihood ratio was 0.19 (95% confidence interval, 0.05-0.67).Elevated plasma FLT3-L in patients who previously received chemotherapy is a predictive measure of the stage of recovery of the bone marrow compartment. FLT3-L seems to identify the likelihood that the patient will experience Gradeor = 3 thrombocytopenia if additional cytotoxic therapy is administered. Knowledge of bone marrow activity should permit therapy that is more aggressive by establishing the earliest possible time for dosing with any cytotoxic agent for which myelosuppression is the dose-limiting toxicity.

We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type.

Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14). All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts. Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2. Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process. Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media. A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity. We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 microM methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures. The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.

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.

The goal of this work was to test whether an antibody-based agent approved for use as a single-photon-emitting imaging agent when radiolabeled with technetium-99m could be labeled comparably with a positron-emitting nuclide, technetium-94m. "Instant kits" containing lyophilized NP-4 antibody Fab' fragment of an anticarcinoembryonic antigen IgG (CEA-Scan) from the same manufactured lot were reconstituted with either Tc-99m or Tc-94m, as solutions of sodium pertechnetate in isotonic saline solution. Radioanalyses of the labeled Fab' fragments by size-exclusion high-performance chromatography and TLC were carried out. Equivalent results were obtained for radioimmunoconjugates when each was analyzed with both methods. Facile incorporation of Tc-94m into tumor-targeting Fab' antibody fragments will enable investigation of such agents for tumor-specific imaging using positron emission tomography.

Multiple myeloma (MM) is the second most common hematological cancer in the United States. It is typically incurable, even with myeloablative chemotherapy and stem-cell transplantation. The epithelial mucin-1 (MUC1) glycoprotein is expressed by normal and malignant epithelial cells but has also been shown to be expressed by MM cells. MUC1 is a useful antigenic target in solid tumors for clinical diagnostic and therapeutic monoclonal antibody (mAb)-based approaches. The MA5 mAb, as well as other anti-MUC1 mAbs reactive with the MUC1 variable number tandem repeat domain, exhibited moderate to strong reactivity with both MM cell lines and clinical samples. To explore the biochemical nature and potential of MUC1 as an antigenic target in MM, studies were performed to: (a) compare the mRNA and the MUC1 glycoprotein species between epithelial cancer and MM cell lines; and (b) develop and use a human MM tumor xenograft model system to study the biodistribution of the MA5 mAb. MA5 mAb was strongly reactive with six of eight human MM cell lines by flow cytometry. In seven of eight MM patient samples (bone marrow and/or peripheral blood) reactivity was found in 10-90% of the cells, whereas normal control (n = 5) and leukemia and lymphoma (n = 5) cells showed only 0-6% reactivity. 125I-labeled MA5 whole-cell binding studies showed quantitatively similar amounts of binding between strongly positive MM lines and high-MUC1-expressing breast carcinoma lines. mRNA expression was assessed by Northern blotting and reverse transcription-PCR. MM cell lines were positive by both methods, with strong similarity in the sizes of the mRNAs and cDNAs that were obtained. Finally, biodistribution experiments were carried out with 131I-labeled MA5 versus a nonbinding control 125I-labeled mAb in a s.c. MM xenograft model. Selective MM tumor uptake of the MA5 mAb was demonstrated, with a potential for delivering a tumor radiation absorbed dose of 8540 cGy/mCi of injected dose compared with 3099 cGy/mCi of tumor-absorbed dose delivered by nonspecific antibody.

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.

To increase tumor-to-nontumor localization ratios of injected radiolabeled antibodies (Abs), several interrelated methods were used.The model systems used were two human carcinoma xenografts grown in nude mice, targeted by antibodies RS11 (antiepithelial glycoprotein-2) or MN-14 (anticarcinoembryonic antigen). The Abs were conjugated with biotin and 111In-benzyl diethylenetriamine pentaacetic acid, and, at various times after injection, were cleared by intraperitoneal injection of galactosylated streptavidin, which delivers the complexes to hepatocytes. The radiolabel used was selected because it is retained within tumors after catabolism of the Ab by the tumor cell but is quite rapidly excreted from hepatocytes into bile.With blood clearance induced at 24 h, and dissection 5 h later, high tumor-to-nontumor ratios were attained. Depending on the model used, tumor-to-blood ratios were 16:1 to 31:1, and tumor-to-nontumor ratios for the kidney, lungs and bone were also high and greatly increased by the clearance regimen. Despite clearance into the liver, tumor-to-liver ratios remained1, due to fairly rapid biliary excretion of the label. The absolute antibody uptake by the tumors was also high, because 24 h was allowed for the Ab to penetrate and bind to cells within the subcutaneous tumors.The method described produced high tumor-to-nontumor ratios at 1 d after injection and may be advantageous for tumor imaging with antibodies. Radiation dosimetry calculations indicate that there is only a slight advantage with this approach for radioimmunotherapy.

We investigated the in vitro cytotoxic potential of Auger electron-emitting radionuclides delivered to the cytoplasm or, more specifically, to lysosomes, via antibodies. The antibody (Ab) used was LL1, which is specific for CD74, an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface. It is taken up in large amounts, approximately 10(7) Ab molecules per cell per day, and delivered to lysosomes. The radioisotopes tested included (111)In, 99mTc and 125I. With sufficient specific activity, approximately 10 mCi/mg Ab, all of these isotopes were potent cytotoxic agents. 125I was active only if a "residualizing" form was used, meaning a form that is trapped within cells after catabolism of the Ab to which it was conjugated (conventional oxidative iodination produces a non-residualizing label). The conjugates of (111)In and 99mTc used are known to be residualizing. One hundred percent cell kill in vitro was obtained with (111)In and 125I, under conditions in which a non-reactive control Ab, conjugated in the same way, produced no significant toxicity. 99mTc was also potent and specific, but appeared somewhat less active than the other isotopes under the conditions evaluated. Although few Abs are accreted by cells at the same rate as LL1, it may be possible to use other Abs to deliver similar amounts of radioactivity, if Abs with higher specific activity can be produced. Such conjugated radioisotopes may be useful for attacking tumor cells in vivo, particularly for single cells or micrometastases.

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.

Mu-9 is a murine monoclonal antibody that is directed against affinity-purified colon-specific antigen-p (CSAp). CSAp is a tumor-associated antigen that is present in 60% of colorectal carcinomas. Preclinical and clinical studies have shown Mu-9 to have excellent targeting abilities. However, as administration of the murine immunoglobulin G (IgG) provoked a human anti-mouse antibody response, chimerization of Mu-9 is warranted for decreasing immunogenicity.Polymerase chain reaction and cDNA library screening methods were used for the cloning of Mu-9 heavy and light chain variable regions for the construction of chimeric Mu-9.The functional chimeric Mu-9 antibody binds to the CSAp antigen in the GW-39 extracts. It has immunochemical properties similar to that of murine Mu-9.The V-region sequence information will be used for design of humanized Mu-9, which will be evaluated for targeting gastrointestinal carcinomas.

The contributions of nuclear imaging tests to the management of cancer are expanding, especially in terms of detection (staging and recurrence), diagnosis, and qualification of patients for certain forms of therapy, and particularly with regard to the tests' ability to identify tumors on a functional basis.This article is a selective review of the advances and clinical management applications of cancer imaging with radiolabeled antibodies (radioimmunodetection or immunoscintigraphy), with the objective of demonstrating how this new imaging technology can contribute to oncologic practice. The contribution of another functional imaging modality, positron emission tomography (PET), also is discussed.Radioimmunodetection and PET imaging have shown particular management contributions in patients with colorectal, breast, or lung carcinoma, as well as in patients with non-Hodgkin's lymphoma (NHL). Two antibody imaging agents, OncoScint and CEA-Scan, currently are available for the detection of colorectal carcinoma, and have shown particular application for disease staging and disclosure of occult lesions. Each of these agents has different characteristics and potential applications. Recent results with CEA-Scan in patients with mammary carcinoma suggest that it may be a useful complementary tool to screening mammography by adding very high specificity. In patients with lung carcinoma, technetium-99m-labeled NR-LU-10 antibody has been shown to be useful in preoperative staging. LymphoScan is under investigation as a staging agent and for revealing residual disease in patients with NHL; it also provides, with a different radioisotope attached and in a humanized antibody form, a therapeutic option for patients with NHL. Similar results can be shown for PET, but this method may not provide the specificity of an antibody-based functional imaging agent, although PET may have a higher lesion sensitivity.Both radioimmunodetection and PET provide new exciting opportunities for earlier and more sensitive functional imaging tests for cancer. In contrast to PET, antibody agents also can serve to qualify a patient as a candidate for an antibody-based therapy.

LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2.The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed.By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen.By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Tumor lesions in the millimeter (mm) range may escape detection with nuclear medicine imaging methods (including single photon emission computed tomography [SPECT]) using radiolabeled monoclonal antibodies (MoAbs). We hypothesized that these lesions still could receive a potentially therapeutic radiation absorbed dose, and therefore should be treated, despite the lack of detection.To simulate this situation, 2-mm beads (0.004 mL) containing approximately 1.15 microCi of iodine-131 (131I) were used. The beads were placed centrally in a 1200-mL liver phantom containing approximately 3 mCi of 131I. The resultant activity concentration on the beads was approximately 288 microCi/mL compared with approximately 2.5 microCi/mL in the phantom, corresponding to a maximum tumor uptake of approximately 0.3% injected dose per gram (%ID/g) if 100 mCi of 131I-labeled immunoglobulin G were administered. The phantom, containing the beads, was imaged by both planar and SPECT techniques at hypothetical Day 1 (time of maximum tumor uptake) and at hypothetical Day 7 to examine the improved target-to-nontarget ratio over time. In addition to imaging the beads, the radiation absorbed dose to the simulated lesions from the beta component emissions of 131I was calculated using absorbed fractions based on Berger's point kernels.Regardless of the conditions used, the beads could not be observed by either planar or SPECT imaging. However, the radiation-absorbed dose to the simulated lesion was calculated to be as high as approximately 6200 centigray (cGy), with an average dose rate of approximately 89.5 cGy/hour.This simulation demonstrates that a relatively high absorbed dose and dose rate can be delivered to mm-sized lesions not observed by conventional nuclear imaging methods, and that these lesions should be considered for radioimmunotherapy with 1311 MoAbs. However, for micrometastases of1 mm, other radionuclides with shorter path length beta particles than 131I, Auger electrons, or alpha particles should be considered.

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.

Single doses of radioantibody are effective at treating single cells or small clusters of cancer cells. However, large tumor masses require either multiple doses of radioantibody or a multimodal approach to therapy using two or more therapeutic agents. Timing of the second dose in a multiple cycle scheme or the second treatment in a multimodal protocol will depend on recovery from toxicity associated with the first treatment.BALB/c mice were dosed with a maximal tolerated dose (MTD) of I-131-MN-14 anti-carcinoembryonic antigen immunoglobulin G (IgG) (250 microCi) or F(ab')2 (1.2 mCi). Mice were redosed with the MTD at one of four time points, either Day 28, 35, 42, or 49 after IgG or Day 14, 21, 28, or 35 after F(ab')2. Survival was monitored to determine the earliest time to redose without lethality. Several studies were then performed to identify an accurate measure of true myelorecovery. Mice were bled retroorbitally on the day of the first dose and at weekly intervals thereafter. Total peripheral white blood cell counts, granulocyte counts, and lymphocyte counts were determined for each animal. GR-1hi expression (percentage of positive cells) and mean channel florescence were determined by FACScan analysis of a blood sample incubated with fluorescein isothiocyanate-anti-mouse Ly-6G (GR-1). In other studies, two mice were killed weekly from a group treated with a single MTD of radioantibody. The weights of their spleens and thymus glands were determined. At that time, femoral marrow was collected from these animals and plated in Methocult M3430 methylcellulose medium (Stemcell Technologies, Vancouver, Canada), and total colony-forming cells in culture were determined. Another population of mice was used to assess normal tissue metabolic activity following radioantibody therapy by quantitating the 4-hour utilization of I-125-dUrd.The ability to redose mice with a second MTD of 1-131-IgG or F(ab')2 required 49 days and 35 days, respectively. Granulocyte and lymphocyte counts did not accurately predict myelorecovery from the first dose. Hematopoietic tissue weight, tissue metabolic activity, and marrow colony forming cells all suggested that redosing was possible 1-2 weeks before it could actually be done without lethality. Percent of cells expressing GR-1hi (60%) and absolute numbers of GR-1hi cells (1400 cells/mm3) suggested myelorecovery in most animals. A greater degree of accuracy was achieved when trends in GR-1hi expression were noted over 2 or more weeks (i.e., the absolute amount of GR-1hi had to exceed levels in untreated mice, as evidence that the hyperproliferative phase of recovery was complete).The only approach that accurately predicted the ability to retreat with myelosuppressive therapy without risk of lethality was an increase in GR-1hi-positive cells above untreated levels. Other approaches are currently being investigated, including the expression of proliferation antigens (e.g., proliferating cell nuclear antigen and Ki-67) in both murine and human samples and differentiation antigens (CD33 and CD34) 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.