Hypothesis: ligand/receptor-assisted nuclear translocation of STATs.

The STAT transcription factors are mediators of signal transduction of a variety of factors, including interferons (IFNs), interleukins, growth factors, and peptide hormones. Subsequent to activation, STATs are translocated to the nucleus apparently through the well-described importin/Ran system, where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, thus raising the question of how they are imported into the nucleus. Of considerable interest is the observation that ligands and/or receptors that signal through STATs contain putative NLSs and, where examined, either ligand or receptor undergoes nuclear translocation. We hypothesize that ligands and/or their receptors serve as vehicles for the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFNgamma as a model system, we provide a possible mechanism for how this direct role is fulfilled. A functional NLS has been identified in a C-terminal domain of IFNgamma. This domain and the NLS contained within are crucial for the biological properties of IFNgamma in that a peptide encompassing this domain is sufficient to induce an antiviral state. Further, this domain binds specifically to a membrane-proximal region internal cytoplasmic domain of the alpha subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

Cytokine-receptor complexes as chaperones for nuclear translocation of signal transducers.

A variety of ligands that include interleukins, interferons, and growth hormones activate STAT transcriptions factors. When activated, STATs are translocated to the nucleus apparently through the well described importin/Ran system where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, raising the question of how they are imported into the nucleus. Surprisingly, most ligands and/or receptors that signal through STATs contain putative NLSs, and where examined either ligand or receptor undergo nuclear translocation. We hypothesize that these ligands and/or their receptors serve as chaperones in the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFN gamma as a model system we provide a possible mechanism for how this direct role is fulfilled. A C-terminal domain of IFN gamma has been identified that contains a functional NLS. Besides the fact that this domain, and the NLS in particular, is crucial for the biological properties of IFN gamma, a peptide encompassing this domain is sufficient to induce an antiviral state. Moreover, this domain interacts exclusively with an internal cytoplasmic domain of a subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the elements of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

Phase I trial of iodine 131-labeled COL-1 in patients with gastrointestinal malignancies: influence of serum carcinoembryonic antigen and tumor bulk on pharmacokinetics.

PURPOSE: COL-1 is a high-affinity murine monoclonal antibody (MAb) specific for carcinoembryonic antigen (CEA). A phase I trial was conducted in which a uniform quantity of antibody labeled with escalating doses of iodine 131 (131I) was administered to patients with advanced gastrointestinal (GI) malignancies to evaluate tolerance and pharmacokinetics. PATIENTS AND METHODS: Eighteen patients with advanced, assessable GI malignancies (16 colon, one pancreas, and one gastric) previously treated with conventional chemotherapy (but no pelvic radiation) received 20 mg of COL-1 labeled with 131I, with doses from 10 mCi/m2 to 75 mCi/m2. In this cohort, the baseline serum CEA level ranged from 6 to 2,739 ng/mL (mean +/- SD, 500 +/- 639). RESULTS: Nuclear imaging detected at least one tumor site in all 18 patients; 82% of all tumor involved organs were positive and 58% of all lesions > or = 1.0 cm were detected. Immune complexes were detected in 89% of patients 5 minutes after completion of infusion, and levels correlated with CEA levels (r = .71). Elevated CEA (> 500 ng/mL) and tumor bulk (total tumor area > 150 cm2) correlated directly with clearance of serum radioactivity and inversely with serum half-life and cumulative serum radioactivity parameters. Nonhematologic toxicity was mild and non-dose-limiting. Hematologic toxicity, particularly thrombocytopenia, was both dose-related and dose-limiting. The maximal-tolerated dose is 65 mCi/m2. The correlation between dose (millicuries per square meter) and thrombocytopenia was made stronger, by accounting for either variation in pharmacokinetics, or variation in serum CEA and tumor bulk. CONCLUSION: 131I-COL-1 is well tolerated, except for hematologic toxicity. These data suggest that patients with highly elevated circulating CEA levels and/or increased tumor bulk may clear 131I-labeled COL-1 more rapidly from the circulation and experience less myelosuppression.

Phase I radioimmunotherapy trial with iodine-131-CC49 in metastatic colon carcinoma.

UNLABELLED: CC49 is a murine monoclonal antibody (MAb) that reacts against the TAG-72 antigen. We carried out a Phase I study with escalating doses of 131I-CC49 in patients with advanced colorectal cancer expressing the TAG-72 antigen to determine the dose-limiting toxicity and therapeutic efficacy, if any, of the radioimmunoconjugate. METHODS: Twenty-four patients with TAG-72- expressing colorectal cancer were treated with escalating doses of 131I-CC49 starting at 15 mCi/m2 and going up to 90 mCi/m2 of 131I labeled to 20 mg MAb CC49. Patients were selected if TAG-72 was expressed in > or = 50% of cells in previously resected tumor and at least one metastasis was demonstratable on standard imaging such as CT. All patients had failed conventional chemotherapy and had not received prior radiotherapy or murine MAb. Patients were under radiation isolation precautions until whole-body radioactivity decreased to < or = 5 mR/hr at 1 m. Whole-body scintigrams were obtained prior to discharge and 1 and 2 wk after infusion in all patients. SPECT imaging was carried out at least once in all patients. RESULTS: All patients had excellent targeting of radioactivity to known tumor sites. There was no nonhematologic toxicity. Hematologic toxicity was more pronounced in those patients who had received extensive prior chemotherapy. There were no major responses. All patients developed an immune response (HAMA) within 4 wk of therapy. CONCLUSION: Radioimmunotherapy with 131I-CC49 is safe and there is significant therapeutic efficacy in this Phase I trial at the doses studied. There is excellent targeting of radioactivity to antigen-positive tumors. Dose-limiting toxicity is hematopoietic, with the maximum tolerated dose in this group of heavily pretreated patients being 75 mCi/m2.

Biodistribution and preclinical radioimmunotherapy studies using radiolanthanide-labeled immunoconjugates.

Lutetium-177 (177Lu), samarium-153 (153Sm), and yttrium-90 (90Y) are members of the family of elements known as lanthanides or rare earths. Monoclonal antibody CC49, a murine immunoglobulin (Ig) G1, which is reactive with the tumor-associated antigen TAG-72, previously has been shown to react with a wide range of human carcinomas. The authors review here the comparative biodistributions of CC49 IgG and F(ab')2 fragments labeled with 177Lu, 153Sm, and 90Y using the bifunctional chelating agent PA-DOTA. The authors also review the results of a biodistribution study comparing iodine-125-labeled and 177Lu-labeled CC49 sFv, and the use of 177Lu-CC+9 IgG in an experimental therapy model. Chelation and conjugations gave similar yields, and the labeled proteins showed similar retention of immunoreactivity regardless of the isotope used for both IgG and F(ab')2. Biodistribution data obtained in athymic mice bearing LS-174T human colon carcinoma xenografts likewise showed no differences among the three radioisotopes for both IgG and F(ab')2. Femur uptake of radioactivity was lower than previously reported for other radiolanthanide immunoconjugates. Different metabolic patterns were observed for radioiodinated versus radiometal-labeled sFv, particularly in the kidney, where localization of the latter was increased dramatically. 177Lu-CC49 was found to delay the growth of established LS-174T human colon carcinomas in athymic mice at a single dose of 50 microCi. Elimination of established tumors was demonstrated over the observation period (77 days) using single administrations of 200 or 350 microCi. Dose fractionation experiments revealed that the mice tolerated 750 microCi (3 x 250 microCi, given weekly), whereas > 50% of the mice died after receiving a single administration of approximately 500 microCi. In isotype-matched control experiments, a large differential in the therapeutic effects was observed between 177Lu-labeled control antibody and CC49.

Clinical comparison of radiolocalization of two monoclonal antibodies (mAbs) against the TAG-72 antigen.

Ten patients with colorectal cancer metastases received 125I-B72.3 and 131I-CC49 prior to laparotomy (five patients received 1 mg, and five 20 mg of each mAb). Tumor:serum ratios of 131I-CC49 were better than those of 125I-B72.3 (P < 0.01 at 1 mg; P = 0.05 at 20 mg; P < 0.01 at both doses). All known lesions > or = 1 cm in diameter were visualized at the 20 mg dose. There was no difference in absolute tumor uptake of 125I-B72.3 or 131I-CC49. We conclude that mAb CC49 has better relative uptake in colorectal cancers than mAb B72.3.

Therapeutic efficacy of a high-affinity anticarcinoembryonic antigen monoclonal antibody (COL-1).

COL-1 is a murine IgG2a monoclonal antibody (MAb) with a high affinity (1.4 x 10(9) M-1) for carcinoembryonic antigen (CEA) and no detectable reactivity for CEA-related antigens, such as nonspecific cross-reacting antigen (NCA) and normal fecal antigen (NFA). 125I-labeled COL-1 IgG was shown to efficiently and specifically target the LS-174T human colon carcinoma xenograft in athymic mice. Dose titration studies in this same model with 131I-labeled COL-1 demonstrated reduction of tumor growth rate when 300 microCi of the immunoconjugate was used (0.005 > p > 0.001). Administration of higher levels as a single dose led to increased toxicity. Dose fractionation experiments with 131I-COL-1 demonstrated the ability to administer much higher levels of the immunoconjugate with little or no toxicity, which resulted in a greater therapeutic efficacy. For example, three fractions of 200 microCi of 131I-COL-1 given at weekly intervals (for a total of 600 microCi) resulted in the substantial reduction (p < 0.0005) of the growth of established tumors in 100% (7/7) of mice, and in no evidence of tumor growth in 71% (5/7) of mice, at the end of the 63-day observation period. These results thus demonstrate the potential therapeutic efficacy for radiolabeled COL-1 in clinical trials and demonstrate the principle of the advantage of dose fractionation protocols for this immunoconjugate.

Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates.

The effect of the relative affinity (Ka) on the antitumor efficacy of monoclonal antibodies (MAbs) has been questioned. It has previously been shown in experimental models that the use of MAbs with higher relative Kas manifests itself in a higher percentage of injected dose of MAb bound to tumor. On the other hand, mathematical models have proposed that the use of higher affinity MAbs may be disadvantageous for antitumor effects, since higher Ka MAbs would bind more antigen and prevent penetration of MAb through tumor. To test this hypothesis, three MAbs reacting to the human pancarcinoma antigen TAG-72 were used as radioimmunoconjugates for therapeutic efficacy versus the LS-174T human colon carcinoma xenograft. MAbs B72.3, CC49, and CC83 have all been shown by depletion studies to react to the same molecule and to all react with overlapping epitopes. While the relative Ka of B72.3 is 2.5 x 10(9) M-1, the relative Kas of CC49 and CC83 are 16.2 and 27.7 x 10(9) M-1, respectively. Each MAb was radiolabeled with 131I, and each radioimmunoconjugate was assayed at five dose levels for therapeutic efficacy using the human xenograft model. The results of these studies demonstrate substantial therapeutic advantage of the higher affinity MAbs CC49 and CC83 versus B72.3 at every dose level. While 500 microCi of B72.3 were required to reduce tumor growth in only a minority of tumor-bearing animals, the use of the same amount or less of the radioimmunoconjugates of CC49 or CC83 resulted in strong antitumor effects in 80 to 100% of tumor-bearing animals. Thus, stronger antitumor effects were seen using as little as 2.5- to 3-fold less of the higher Ka immunoconjugates CC49 and CC83 as compared with B72.3. While we acknowledge the potential disadvantages of higher Ka MAbs in some situations, at least the experimental studies and model system described here show that a distinct therapeutic advantage exists with the use of higher affinity immunoconjugates.

Monoclonal antibody-based therapy of a human tumor xenograft with a 177lutetium-labeled immunoconjugate.

177Lutetium (177Lu) is a member of the family of elements known as lanthanides or rare earths. Monoclonal antibody (MAb) CC49, a murine IgG1, which is reactive with the tumor-associated antigen, TAG-72, has been shown previously to react with a wide range of human carcinomas; CC49 reacts to a different epitope on the TAG-72 molecule than MAb B72.3 and has a higher binding affinity. We report here the first use of a 177Lu-labeled immunoconjugate, 177Lu-CC49, in an experimental therapy model for human carcinoma. 177Lu-CC49 was shown to delay the growth of established LS-174T human colon carcinomas in athymic mice at a single dose of 50 microCi. Overt toxicity was observed with the administration of approximately 500 microCi of 177Lu-CC49 in which 5 of 9 mice died of apparent marrow toxicity. A single administration of 200 or 350 microCi of 177Lu-CC49, however, was shown to eliminate established tumors through the 77-day observation period after MAb administration. Dose fractionation experiments revealed that at least 750 microCi of 177Lu-CC49 (250 microCi/week for 3 consecutive weeks) was well tolerated in that 9 of 10 mice survived. Moreover, this dose schedule was able to eliminate the growth of relatively large (300 mm3) human colon tumor xenografts in 90% of the animals treated. Single-dose and dose fractionation studies were also carried out with an isotype-matched control MAb, 177Lu-MOPC-21. In all dose schedules, a large differential was seen between the therapeutic effects of the 177Lu-CC49 versus that of the 177Lu-control MAb. The merits and limitations of the use of 177Lu-labeled immunoconjugates (in particular, 177Lu-CC49) are discussed in terms of potential novel therapeutics for human carcinoma.

Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy.

Monoclonal antibody (MAb) B72.3 IgG was radiolabeled with 131I and administered to female athymic NCr-nu mice bearing the LS-174T human colon adenocarcinoma xenograft to determine if fractionation of MAb dose had any advantage in tumor therapy. In the LS-174T xenograft, only approximately 30%-60% of tumor cells express the B72.3-reactive TAG-72 antigen. The LS-174T xenograft was used to reflect the heterogeneity of the TAG-72 antigen often seen in biopsy specimens from patients. In contrast to a single 600-muCi dose of 131I-B72.3 IgG where 60% of the animals died from toxic effects, two 300-muCi doses of 131I-B72.3 IgG (total of 600 muCi) reduced or eliminated tumor growth in 90% of mice, with only 10% of the animals dying from toxic effects. Dose fractionation even permitted escalation of the dose to three doses (each 1 wk apart) of 300 muCi of 131I-B72.3 IgG (for a total of 900 muCi), resulting in even more extensive tumor reduction or elimination and minimal toxic effects. The use of an isotype-matched control MAb revealed a nonspecific component to tumor growth retardation, but the use of the specific B72.3 IgG demonstrated a much greater therapeutic effect. Tumors that had escaped MAb therapy were analyzed for expression of the B72.3-reactive TAG-72 antigen with the use of the immunoperoxidase method; they were shown to have the same antigenic phenotype as the untreated tumors. We verified tumor elimination by killing the test animals after a 7-week observation period and performing histologic examination of tumor sites. We also monitored toxic effects by histologic examination of numerous organs, including bone marrow. These studies thus demonstrate the advantage of dose fractionation of a radiolabeled MAb for tumor therapy. We anticipate that the concept of dose fractionation can be practically applied in radioimmunotherapeutic clinical trials with the development and use of recombinant-chimeric MAbs and modified constructs.

Binding of radiolabeled MAb B72.3 administered intravenously and intraperitoneally in colorectal cancer patients. An overview.

Monoclonal antibody (MAb) B72.3 coupled to 131I is currently being used in clinical trials for the diagnostic imaging of gastrointestinal, ovarian, and breast carcinomas. The MAb has also been used extensively in immunohistochemical studies and in the development of radioimmunoassays to define antigen levels in tissues and sera of cancer patients. When administered intravenously, the radionuclide-conjugated MAb has localized tumor in over 70 percent of patients. When intraperitoneal administration was studied, the MAb-radionuclide conjugate successfully imaged peritoneal lesions; moreover, in 3 of 10 patients, B72.3 detected lesions not found by conventional methods. Concomitant administration of 131I labeled B72.3 intraperitoneally and 125I labeled B72.3 intravenously showed that the intraperitoneal route localized peritoneal implants at least two times better than the intravenously administered MAb. Conversely, hematogenously borne or non-implant lesions were better localized with intravenously administered B72.3.

Radiolabeled monoclonal antibody B72.3 localization in metastatic lesions of colorectal cancer patients.

We have previously demonstrated a high degree of selective binding of monoclonal antibody (MAb) B72.3 to carcinomas of the colon, ovary, and breast in contrast to normal adult tissues using in vitro assays. In this report we demonstrate selective tumor localization in colorectal cancer patients after intravenously administering 131I-labeled MAb B72.3 IgG. Radiolocalization Indices (RI) (i.e. cpm 131I-labeled MAb per gram of tumor vs cpm per gram of normal tissues), were obtained by direct analyses of biopsy materials. Using an RI of greater than or equal to 3 as a positive localization, tumor lesions in various sites from 17/20 patients scored positive. In eight of these patients, all tumor lesions demonstrated RIs of greater than 3, while in five patients RIs of some lesions were greater than 10 and as high as 30-46. Seventy percent (99/142) of the tumor lesions showed RIs of greater than 3, while only 12 of 210 histologically confirmed normal tissues examined showed RIs of greater than 3. These tissues were either adjacent to the tumor or the draining tumor masses or, as in the case of two patients, was caused by high levels of circulating immune complexes that deposited in the spleen. Positive scintigraphic images (confirmed at surgery) were observed in 14/27 patients. No toxicity or adverse reactions were observed with either MAb. These studies provide absolute quantitative analyses of the actual delivery of radiolabeled MAb to carcinoma lesions vs a wide range of adjacent and distal normal tissues and establishes the means for other diagnostic and potential therapeutic applications of this antibody alone, or in combinations with other monoclonal antibodies.

Lymphomas and leukemias in the relatives of patients with mycosis fungoides.

Of 526 consecutive patients with cutaneous T-cell lymphomas, 21 had first-degree relatives with lymphoproliferative or hematopoietic malignancies. Twenty-nine such tumors occurred in the 21 kindreds. Hodgkin's disease accounted for one-third of this total, with various leukemias (11 cases), non-Hodgkin's lymphoma (five cases), and multiple myeloma (three cases) comprising the remainder. These data suggest that genetically-determined immunoregulatory abnormalities may represent a shared pathway of oncogenesis in diverse lymphoproliferative and hematopoietic malignancies.

Evaluation of emergency ambulance characteristics under several criteria.

A methodology and analysis are presented for evaluating response time characteristics of emergency ambulance systems. The methodology is based on a Monte Carlo simulation technique and a heuristic optimal-seeking technique for locating emergency ambulances under several criteria based on response time distribution. Optimization criteria include minimum mean system response time, minimum system fractile response time and minimum level-loaded response time. The evaluation methodology is applied to the metropolitan area of Los Angeles County. Ambulance response characteristics and loads are discussed in detail. From these results alternative dispatch polices can be evaluated. Complementing the analysis is a presentation of a sensitivity analysis and an analysis of existing ambulance sites. Unique to the methodology is the adaption of the heuristic optimal-seeking technique to include any of the three criteria and the effectiveness of the methodology for analyzing small or large ambulance systems.

Response of the rat erythrocyte to ozone exposure.

Sprague-Dawley rats were exposed to high (6--8 ppm) and moderate (1.5 ppm) amounts of ozone (O3) for various time periods. Response of the rat erythrocyte to ozone was monitored with red blood cell potassium (rubidium) influx studies, with storage stress combined with ultrastructural studies and with levels of erythrocyte glutathione peroxidase and superoxide dismutase. Erythrocytes of rats exposed to O3 showed no significant changes either in their potassium influx or in their glutathione peroxidase and superoxide dismutase activities compared to controls. Erythrocyte differential counts on O3-exposed animals showed significant changes initially as well as following storage stress compared to controls. Rats exposed to 8 ppm O3 for 4 h showed a marked increase in echinocytes. These consistent transformations from discocytes to echinocytes following O3 exposure suggest latent erythrocyte damage has occurred.

Response of the iron-deficient erythrocyte in the rat to hyperoxia.

Normal and iron-deficient rats were exposed to 90% O2 at 760 Torr for 24 or 48 h. Erythrocyte response to hyperoxia was monitored by potassium (rubidium) influx studies, by storage stress, and by ultrastructural studies. Normal rat erythrocytes exhibited morphological changes and decrease of ouabain-sensitive potassium influx compared to unexposed controls. Both components of erythrocyte potassium influx were affected by iron deficiency. Erythrocytes from unexposed iron-deficient rats showed a 50% increase in ouabain-sensitive potassium influx compared to controls. Iron-deficient rats exposed to hyperoxia for 24 or 48 h, had erythrocytes with morphological changes. Erythrocytes of iron-deficient rats exposed for 24 h showed no influx change; those exposed for 48 h showed a decrease of ouabain-sensitive influx compared to erythrocytes of controls.