[18F]FDG and [18F]FLT uptake in human breast cancer cells in relation to the effects of chemotherapy: an in vitro study.

Increased 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG) uptake is the most commonly used marker for positron emission tomography in oncology. However, a proliferation tracer such as 3'-deoxy-3'-[18F]fluorothymidine (FLT) might be more specific for cancer. 3'-deoxy-3'-[18F]fluorothymidine uptake is dependent on thymidine kinase 1 (TK) activity, but the effects of chemotherapeutic agents are unknown. The aim of this study was to characterise FDG and FLT uptake mechanisms in vitro before and after exposure to chemotherapeutic agents. The effects of 5-fluorouracil (5-FU), doxorubicin and paclitaxel on FDG and FLT uptake were measured in MDA MB231 human breast cancer cells in relation to cell cycle distribution, expression and enzyme activity of TK-1. At IC50 concentrations, 5-FU resulted in accumulation in the G1 phase, but doxorubicin and paclitaxel induced a G2/M accumulation. Compared with untreated cells, 5-FU and doxorubicin increased TK-1 levels by >300. At 72 h, 5-FU decreased FDG uptake by 50% and FLT uptake by 54%, whereas doxorubicin increased FDG and FLT uptake by 71 and 173%, respectively. Paclitaxel increased FDG uptake with >100% after 48 h, whereas FLT uptake hardly changed. In conclusion, various chemotherapeutic agents, commonly used in the treatment of breast cancer, have different effects on the time course of uptake of both FDG and FLT in vitro. This might have implications for interpretation of clinical findings.

Hexokinase III, cyclin A and galectin-3 are overexpressed in malignant follicular thyroid nodules.

BACKGROUND: Distinguishing malignant thyroid nodules in patients with follicular cytology by fine-needle aspiration (FNA) remains problematic. The large majority of thyroid nodules (> 85%) are overtreated. Therefore, a clear need exists to develop more accurate initial diagnostic tests for follicular thyroid nodules. Galectin-3 is the most recent promising marker to aid discrimination between benign and malignant thyroid lesions; however, this biomarker can be absent in follicular malignancies. AIMS: This study was undertaken to determine whether additional biomarkers can help to discriminate between benign and malignant thyroid nodules. METHODS: Surgical specimens of 36 patients with benign (n = 12) and malignant (n = 24) thyroid nodules showing follicular cytology were assessed by immunohistochemistry for the expression of galectin-3 and novel biomarkers. RESULTS: Expression of hexokinase III (HK III) (P = 0.000) cyclin A (P = 0.002) and galectin-3 (P = 0.003) differed significantly between benign and malignant thyroid nodules. HK III had a sensitivity of 79% [95% confidence interval (CI) 60-91] and a specificity of 100% (95% CI 76-100) in predicting malignancy. Galectin-3 had a sensitivity of 79% (95% CI 56-91) and a specificity of 75% (95% CI 47-91) in predicting malignancy. Combining HK III, cyclin A and galectin-3 in a parallel test increased the sensitivity to 96% (95% CI 80-99) while the specificity remained at a high level of 75% (95% CI 47-91). Leave-one-out cross-validation demonstrated a stable predictive validity of a model based on HK III, cyclin A and galectin-3. CONCLUSIONS: In this study, we have demonstrated that in addition to galectin-3, HK III and cyclin A profiles could be important biomarkers in predicting malignancy in follicular thyroid nodules. The use of these biomarkers may allow an accurate preoperative diagnosis of thyroid cancer, which can be cost saving and may avoid serious morbidity such as vocal cord paralysis. The value of the suggested biomarkers warrants further evaluation in a large prospective study on cytological samples of follicular thyroid nodules.

Biodistribution of (111)indium-labeled engineered human antibody CTMO1 in ovarian cancer patients: influence of protein dose.

Thirty-one patients suspected of having ovarian cancer received a single i.v. injection of radiolabeled (100 MBq (111)In) engineered human CTMO1 (hCTMO1) to investigate its potential as an internalizing drug carrier. hCTMO1 is a complementary-determining region-grafted human IgG4 monoclonal antibody recognizing an ovarian carcinoma-associated antigen, the MUC-1-gene product. The amount of radioactivity was determined in tumor tissue, various normal tissues, including liver biopsies, and blood samples obtained at laparotomy, 6 days after injection of either 0.1 or 1.0 mg hCTMO1/kg of body weight. Circulating antigen-15-3 was measurable in all patients before injection, and immune complex formation was already present at the end of infusion. In the 0.1 mg/kg group, most of the radioactivity was bound to immune complexes, whereas in the 1.0 mg/kg group, most was bound to IgG monomers. Increasing the hCTMO1 dose 10-fold did not influence the overall disappearance of (111)In from the blood, but the elimination half-life of (111)indium bound to immune complexes was increased 2-fold. Uptake in tumor tissue 6 days postinjection at the 0.1 mg/kg dose was 7.6 times higher (P = 0.0009) than in normal tissue and 2.5 times higher (P = 0.03) than in blood. At the 1.0 mg/kg dose, the uptake in tumor tissue was 14.0 times higher (P = 0.0003) than in normal tissue and 8.1 times higher (P = 0.0007) than in blood. Liver activity was substantial (23.7 +/- 10.5 and 18.3 +/- 6.7% of the injected dose/kg for the 0.1 and 1.0 mg/kg dose group, respectively). These results are superior to those found with other clinically tested anti-MUC-1 gene product antibodies. hCTMO1 seems to be a suitable carrier for cytotoxic agents in ovarian carcinoma patients; the better uptake results and tumor-to-blood ratios are obtained at the higher dose of 1.0 mg hCTMO1/kg body weight.

[18F]fluorodeoxyglucose uptake in recurrent thyroid cancer is related to hexokinase i expression in the primary tumor.

Patients suspected of recurrent differentiated thyroid cancer (DTC) may require "blind" (131)I therapy, with the disadvantage of unpredictable efficacy and toxicity. Alternatively, positron emission tomography (PET) with [(18)F]fluorodeoxyglucose ((18)FDG) can document the recurrence, thereby rationalizing therapeutic options. This study compared (18)FDG uptake in vivo with biomarkers expected to be involved in the underlying biological mechanisms. Additionally, we investigated whether such features were present in the primary tumors. Preoperatively, 19 patients with recurrent DTC underwent PET. (18)FDG uptake was compared with histological and immunohistochemical features in surgical specimens of recurrent and primary tumor. Thirteen of 19 recurrences were positive at PET, and (18)FDG uptake was associated with the expression of hexokinase type I (HK I; P = 0.011). All lesions with HK I overexpression were positive on (18)FDG PET. HK I expression in the original primary tumor and the metastases was similar in 82% (rho = 0.648; P = 0.005). In suspected recurrent thyroid cancer, stratification for (18)FDG PET may benefit from pretest immunohistochemical analysis of HK I of the primary tumor, as HK I negativity indicates a low likelihood of PET positivity.

89Zr-immuno-PET for imaging of long circulating drugs and disease targets: why, how and when to be applied?

Positron emission tomography (PET) with 89Zr-labeled monoclonal antibodies (mAbs) or other targeted vehicles (e.g., peptides, nanoparticles and cells), collectively called "89Zr-immuno-PET", can be used for better understanding of disease targets and the in vivo behavior of targeted drugs. This will become increasingly important in the development of next generation mAbs, which are characterized by high potency and/or multiple binding domains. This review provides practical information for researchers who want to implement 89Zr-immuno-PET for answering their own biological and pathological questions or for steering their own drug development program. An overview is given of the reagents, labeling protocols, quality tests and critical steps to come to high quality 89Zr-conjugates, while possibilities for further improvement are discussed. Since PET has the advantage of allowing quantitative imaging, information is provided about standardization of 89Zr quantification. Issues are summarized for consideration when starting preclinical or clinical 89Zr-immuno-PET studies, to enable at the end unequivocal interpretation of results. Finally, many appealing examples are provided of what can be learned from 89Zr-immuno-PET studies, while future directions are outlined. Most of the current examples are still on the characterization of mAbs in oncology, but the review will show that 89Zr-immuno-PET harbors potential for many kinds of targeted drugs and diseases, as well as for elucidating biological processes.

Experimental and clinical analysis of the characteristics of a chimeric monoclonal antibody, MOv18, reactive with an ovarian cancer-associated antigen.

Monoclonal antibody (Mab) MOv18 preferentially reacts with gynecological carcinomas. We have analyzed the characteristics of murine MOv18 (m-MOv18) and chimeric MOv18 (c-MOv18). We found no differences in affinity and binding to IGROV1 cells between c-MOv18 as IgG and F(ab')2 fragments and m-MOv18. In nude mice bearing IGROV1 xenografts, maximum tumor uptake 6-15 hr after i.v. injection of radiolabeled m-MOv18 IgG, c-MOv18 IgG, c-MOv18 F(ab')2 and a control IgG, 2C7, was 10%, 11%, 3% and 4.5% of the injected dose/g (%ID/g), respectively. M- and c-MOv18 IgG retained this level for several days, while c-MOv18 F(ab')2 and 2C7 cleared rapidly from the tumor. Uptake in normal tissues was low, with the exception of high uptake in kidneys for c-MOv18 F(ab')2. Tumor/blood ratios for c-MOv18 F(ab')2 were sixfold higher than for IgG. Radiation absorbed doses to tumor tissue delivered by 10 microCi iodinated m-MOv18 IgG, c-MOv18 IgG and c-MOv18 F(ab')2 were 39 cGy, 49 cGy and 5 cGy, respectively. A cocktail of 125I-c-MOv18 IgG and 131I-c-MOv18 F(ab')2 injected i.v. into an ovarian cancer patient, localized specifically in the tumor. Ovarian cancer tissue samples obtained 2 days postinjection showed a mean uptake of 1.2 x 10(-3) and 2.7 x 10(-3) %ID/g for c-MOv18 IgG and c-MOv18 F(ab')2, respectively. Results from these in vitro and in vivo experiments indicate that c-MOv18 has promise as a Mab for clinical use.

Optimal quality (131)I-monoclonal antibodies on high-dose labeling in a large reaction volume and temporarily coating the antibody with IODO-GEN.

UNLABELLED: A novel, facile procedure for efficient coupling of high doses of (131)I to monoclonal antibodies (MAbs) was developed with minimal chemical and radiation damage. METHODS: To diminish the radiation and chemical burden during labeling, iodination was performed in a large reaction volume and by temporarily coating the MAb with a minimal amount of IODO-GEN. The MAb was coated by injection of IODO-GEN (dissolved in acetonitrile [MeCN]) into the aqueous MAb solution, and the coating was subsequently removed by addition of ascorbic acid. For chemoprotection before, during, and after PD-10 purification of the (131)I-MAbs, ascorbic acid and human serum albumin were used. The effects of autoradiolysis in the starting (131)I solution were countered by treatment with NaOH and ascorbic acid. For this so-called IODO-GEN-coated MAb method, the sensitive chimeric MAb MOv18 (c-MOv18) and the more robust murine MAbs K928 and E48 were used. The high-dose (131)I-labeled MAbs were characterized for radiochemical purity and MAb integrity by thin-layer chromatography, high-performance liquid chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by phosphor imager quantification. The high-dose (131)I-labeled MAbs were also characterized for immunoreactivity. The radiopharmacokinetics and biodistribution of (131)I-c-MOv18 were analyzed in human tumor-bearing nude mice. For comparison, (131)I-c-MOv18 batches were made using the conventional chloramine-T or IODO-GEN-coated vial method. RESULTS: Conventional high-dose labeling of 5 mg c-MOv18 with 4.4 GBq (131)I resulted in a labeling yield of 60%, a radiochemical purity of 90%, an immunoreactive fraction of 25% (72% being the maximum in the assay used), and the presence of aggregation and degradation products. Using similar amounts of (131)I and MAb in the IODO-GEN-coated MAb method, 85%-89% overall radiochemical yield, at least 99.7% radiochemical purity, and full preservation of MAb integrity and immunoreactivity were achieved. For this labeling, 5 mg MAb were coated with 35 microg IODO-GEN during 3 min in a reaction volume of 6 mL. Also, biodistribution was optimal, and tumor accumulation was superior to that of coinjected (125)I-c-MOv18 labeled according to the conventional IODO-GEN-coated vial method. CONCLUSION: A new, facile, high-dose (131)I-labeling method was developed for production of (131)I-labeled MAbs with optimal quality for use in clinical radioimmunotherapy.

Radioimmunotherapy with intravenously administered 131I-labeled chimeric monoclonal antibody MOv18 in patients with ovarian cancer.

UNLABELLED: We investigated the safety and pharmacokinetics of (131)I-labeled chimeric monoclonal antibody MOv18 ((131)I-c-MOv18 IgG) in patients with ovarian cancer and the estimated radiation dose to cancer-free organs and tumor. METHODS: Three patients were injected intravenously with 3 GBq (131)I-c-MOv18. Toxicity was evaluated according to the World Health Organization toxicity scales. Blood sampling was performed for 12 wk after injection. Whole-body and SPECT imaging was performed frequently. Dose rates were obtained with a portable dose-rate measure. Quantitative activity analysis of several organs was performed with the region-of-interest technique. Absorbed doses were calculated using MIRDOSE3. RESULTS: Transient changes in hematologic profiles were seen in 2 patients. Pancytopenia developed in 1 patient; on analysis, she entered the study probably with exhausted bone marrow reserves. Nonhematologic toxicity was mild. No human antichimeric antibody responses were observed. Mean isolation time was 12 d. The plasma elimination half-life increased almost 3-fold compared with that after tracer doses of c-MOv18. Dosimetry showed mean absorbed doses of 163, 380, 276, 338, 781, and 216 cGy, for whole-body, liver, kidney, spleen, lung, and red marrow, respectively. Tumor-absorbed doses ranged from 600 to 3800 cGy. All patients achieved a stable disease state, as confirmed by CT and carcinoma-associated antigen CA 125, lasting from 2 to >6 mo. CONCLUSION: (131)I-labeled c-MOv18 can safely be given to patients with noncompromised bone marrow reserves and may have therapeutic potential particularly in patients with minimal residual disease.

Distribution of OV-TL 3 and MOv18 in normal and malignant ovarian tissue.

AIMS: To analyse the distribution of OV-TL 3 and MOv18 in normal ovarian tissue to determine which antibody is most suitable for (radio)immunotherapy of ovarian carcinoma. METHODS: The distribution of OV-TL 3 and MOv18 was determined using immunohistochemistry and flow cytometry. RESULTS: Epithelial and other cells in many tissues, and leucocytes in peripheral blood, bone marrow and spleen stained positively with OV-TL 3. The staining pattern of MOv18 in normal tissues was more restricted and was confined to epithelial cells in the lung, kidney, pancreas, salivary gland, ovary, Fallopian tubes, and cervix. Reactivity was also observed with pneumocytes in the lung, tubuli in the kidney, acinar cells in the salivary gland and pancreas, in the placenta, and with Kupffer cells in the liver. The staining pattern of chimeric MOv18 was identical with the murine form. OV-TL 3 and MOv18 reacted with 100% and 98% (45/46) of the 46 tested epithelial ovarian cancers, respectively. In ovarian carcinoma tissue homogeneous staining of epithelial cells was observed with OV-TL 3 and more heterogeneous staining with MOv18. In 12 and nine patients, respectively, a difference in staining intensity for OV-TL 3 and MOv18 was observed between various tumour samples from the same patient. CONCLUSION: MOv18 has greater therapeutic potential because of its restricted reactivity with normal tissues and especially, in contrast to OV-TL 3, its lack of reactivity with haematopoietic cells.

(R)- and (S)-[11C]verapamil as PET-tracers for measuring P-glycoprotein function: in vitro and in vivo evaluation.

The mdr1 gene product P-glycoprotein (P-gp) is involved in the bioavailability and pharmacokinetics of various drugs. Racemic [(11)C]verapamil has been used to image P-gp expression in vivo. A racemic tracer, however, is not suitable for quantification. The purpose of the present study was to identify the most appropriate enantiomer of [(11)C]verapamil as a potential PET-tracer for quantifying P-gp function. The two enantiomers, (R)- and (S)-[(11)C]verapamil, were synthesized and studied in vivo. For the in vivo model mdr1a/1b double gene knock-out and wild type mice were used. The in vitro study made use of the LLC-PK1 MDR cell line to examine the P-gp mediated transport of both enantiomers. The biodistribution of (R)- and (S)-[(11)C]verapamil in dKO and WT mice demonstrated no stereoselectivity of verapamil for P-gp in the blood-brain barrier and in the testes. In addition, no significant differences in P-gp transport for both enantiomers were observed in the in vitro experiments. Previous studies have shown that (R)-verapamil is metabolized less in man and that it has lower affinity for calcium channels. Since (R)- and (S)-verapamil have equal transport for P-gp, the (R)-enantiomer seems to be the best and safest candidate as PET-tracer for measuring P-gp function in vivo.

Use of an in-field-of-view shield to improve count rate performance of the single crystal layer high-resolution research tomograph PET scanner for small animal brain scans.

The count rate performance of the single LSO crystal layer high-resolution research tomograph (HRRT-S) PET scanner is limited by the processing speed of its electronics. Therefore, the feasibility of using an in-field-of-view (in-FOV) shield to improve the noise equivalent count rates (NECR) for small animal brain studies was investigated. The in-FOV shield consists of a lead tube of 12 cm length, 6 cm inner diameter and 9 mm wall thickness. It is large enough to shield the activity in the body of a rat or mouse. First, the effect of this shield on NECR was studied. Secondly, a number of experiments were performed to assess the effects of the shield on the accuracy of transmission scan data and, next, on reconstructed activity distribution in the brain. For activities below 150 MBq NECR improved only by 5-10%. For higher activities NECR maxima of 1.2E4 cps at 200 MBq and 2.2E4 cps at 370 MBq were found without and with shield, respectively. Listmode data taken without shield, however, were corrupted for activities above 75 MBq due to data overrun problems (time tag losses) of the electronics. When the shield was used data overrun was avoided up to activities of 150 MBq. For the unshielded part of the phantom, transmission scan data were the same with and without shield. The estimated scatter contribution was approximately 8.5% without and 5.5% with shield. Reconstructed emission data showed a difference up to 5% in the unshielded part of the phantom at 5 mm or more from the edge of the shielding. Of this 5% about 3% results from the difference in the uncorrected scatter contribution. In conclusion, an in-FOV shield can be used successfully in an HRRT PET scanner to improve NECR and accuracy of small animal brain studies. The latter is especially important when high activities are required for tracers with low brain uptake or when multiple animals are scanned simultaneously.

Pharmacokinetics and biodistribution of a new anti-episialin monoclonal antibody 139H2 in ovarian-cancer-bearing nude mice.

The new murine anti-episialin monoclonal antibody (mAb) 139H2 has been selected for its strong reactivity with a series of human ovarian cancer xenografts. In the present report we describe the characteristics of mAb 139H2 investigated in vitro as well as in vivo. Scatchard plot analysis using the human ovarian cancer cell line NIH:OVCAR-3 showed an affinity constant of 1 x 10(8) M-1 and the expression of 7 x 10(6) antigenic sites/cell. Reactivity with OVCAR-3 xenograft tissue was intense, localized at the cell membrane, heterogeneously distributed, and mainly detectable at the apical site of the cell. Administration of radiolabelled mAb 139H2 to nude mice bearing s.c. OVCAR-3 xenografts showed specific uptake in the tumour up to 9% of the injected dose/g. The maximum uptake in the tumour was retained for 3.5 days and mAb 139H2 cleared from the tumour with a half-life of 5.5 days. The half-life in blood was 50 h and no antibody-antigen complex formation could be detected. Poor uptake and no retention in episialin-negative WiDr colon cancer xenografts demonstrated specificity. Administration of an excess of an unlabelled irrelevant mAb did not influence the uptake in the OVCAR-3 xenografts or in other tissues. In contrast, tumour uptake decreased after addition of 300 micrograms or more unlabelled mAb 139H2 to a tracer dose of radiolabelled mAb 139H2. The uptake of mAb 139H2 in OVCAR-3 xenografts appeared inversely related to the tumour size.

Human ovarian cancer xenografts in nude mice: characterization and analysis of antigen expression.

We have characterized 13 different human ovarian cancer xenografts grown subcutaneously in nude mice. The tumor lines represented 5 histological subtypes: serous (4), mucinous (4), clear-cell carcinoma (1), carcinosarcoma (1) and undifferentiated (3). The specific histology and the degree of differentiation resembled those of the original patients' tumors and were maintained upon serial transfer. Volume doubling times of the xenografts ranged from 3.5 to 15 days. The xenografts were also analyzed for their antigen expression using 20 monoclonal antibodies (MAbs) reactive with 15 tumor-associated antigens. Immunohistochemical examination of tissue sections showed a positive reaction pattern with MAbs 115D8, 140C1, 139H2, 175C5, HMFG1 and HMFG2, each recognizing episialin, as well as with MAbs AUA1, 358.4.32 and 199-157 in xenografts of the serous, mucinous and clear-cell carcinoma subtype. MAb OC125 was reactive with xenografts of the serous subtypes. Other antibodies, such as 494 and OV-TL3 infrequently demonstrated positive reactions. Reactivity of all MAbs was low in the carcinosarcoma and undifferentiated tumor lines. With the exception of AUA1, 495 and 126E5, all MAbs revealed a heterogeneous staining pattern. MAbs against episialin and OC125 predominantly stained the apical site of the tumor cells. Strongest reactivity with almost all histological subtypes was observed with MAbs 115D8, 140C1, 139H2 and AUA1. In cases where we were able to compare the patients' tumor tissue with the respective xenografts, retention of antigen expression was demonstrated in each instance. Release of tumor-associated antigens was shown for CA125 in 2 serous-tumor lines, for CA15.3 in 1 serous-tumor line, and for CEA in 3 lines of the mucinous subtype. This panel of human tumor xenografts could be a valuable tool to determine the potential usefulness of MAb-guided therapy in ovarian cancer.

Influence of dose and schedule on the therapeutic efficacy of 131I-labelled monoclonal antibody 139H2 in a human ovarian cancer xenograft model.

The therapeutic efficacy of various doses and schedules of 131I-labelled anti-episialin monoclonal antibody (MAb) 139H2 was assessed in the NIH:OVCAR-3 human ovarian cancer xenograft model. Radioimmunotherapy was started at the time s.c. tumors were well established (100 to 300 mm3). The anti-tumor effects induced by i.v. injections of 131I-MAb 139H2 were dose- and schedule-dependent. Optimal growth inhibition and long-lasting complete tumor regressions were obtained with 2 injections of 500, 700 or 750 muCi 131I-MAb 139H2 per mouse given with a 2-week interval. The percentage of tumors with more than 50% reduction of their initial volume after treatment with a total dose of 1,000 muCi 131I-MAb 139H2 per mouse, given as 10 injections of 100 muCi (3 x/week), 4 injections of 250 muCi (2 x/week), 10 injections of 100 muCi (5 x/week) within a period of 3 weeks, or 2 injections of 500 muCi with a 2-week interval, was 9%, 40%, 64% and 75% respectively. Unlabelled MAb 139H2 did not affect tumor growth, while the effects of 131I-control MAb were minor and transient. 131I-MAb 139H2 treatment did not select for outgrowth of episialin-negative cells in the OVCAR-3 xenografts. Highest absorbed doses of whole-body-radiation were calculated for 2 injections (500 to 750 muCi 131I-MAb 139H2) given with the 2-week interval. The radiation dose to the tumor after a single injection of 500 muCi 131I-MAb 139H2 was 1,300 cGy over 7 days, which appeared slightly lower than the dose calculated after administration of a tracer dose of iodinated MAb 139H2.

Comparison of 131I-labelled anti-episialin 139H2 with cisplatin, cyclophosphamide or external-beam radiation for anti-tumor efficacy in human ovarian cancer xenografts.

Three human ovarian cancer xenografts of different origin and grown s.c. in nude mice as well-established tumors were studied for their sensitivity to cisplatin (CDDP), cyclophosphamide (CTX), 131I-labelled anti-episialin monoclonal antibody (MAb) 139H2, or external-beam radiotherapy. The maximum tolerated dose of CDDP given weekly i.v. x 2 induced a tumor growth inhibition (GI) of 77.5% and 85.1% of the serous xenografts Ov.Ri(C) and OVCAR-3, respectively. The mucinous xenograft Ov.Pe was relatively resistant to CDDP. The maximum tolerated dose of CTX, given i.p. x 2 with a 2-week interval, induced a GI between 52.9% and 59.7% for each of the 3 xenografts. Radioimmunotherapy with 500-750 microCi 131I-specific MAb 139H2, administered i.v. x 2 with a 2-week interval, was more effective than CDDP or CTX. The 500 microCi 131I-MAb 139H2 schedule induced 100% GI in Ov.Ri(C) xenografts and all tumors were cured. The same schedule was slightly less effective in OVCAR-3 xenografts, but complete tumor regressions could still be obtained. Ov.Pe xenografts were least sensitive to radioimmunotherapy. The 2 injections of 500 microCi 131I-control MAb gave only transient growth inhibition of OVCAR-3 and Ov.Pe tumors, but gave complete regressions of Ov.Ri(C) xenografts. Biodistribution using tracer doses of 131I-MAb 139H2 and 125I-control MAb showed different degrees of specificity for MAb 139H2 in the 3 xenografts. Radiation doses absorbed in OV.Ri(C), OVCAR-3 and Ov.Pe xenografts per 10 microCi injected dose were 30, 41 and 29 cGy respectively. Treatment with 10 Gy external-beam radiation suggested that the effects of radioimmunotherapy in each tumor line were related to the intrinsic radiosensitivity of the xenografts.

Comparison of the pharmacokinetics, biodistribution and dosimetry of monoclonal antibodies OC125, OV-TL 3, and 139H2 as IgG and F(ab')2 fragments in experimental ovarian cancer.

Monoclonal antibody (MAb) 139H2 was previously shown to localise specifically into ovarian cancer xenografts in nude mice. MAb 139H2 was compared with MAbs OC125 and OV-TL 3, all reactive with ovarian carcinomas, for the binding characteristics as IgG and F(ab')2 fragments with the use of the OVCAR-3 cell line grown in vitro and as s.c. xenografts. Immunoperoxidase staining of OVCAR-3 tissue sections with MAbs OC125 and 139H2 was heterogeneous, whereas MAb OV-TL 3 showed homogeneity. No differences in binding were observed between IgG and F(ab')2. The avidity expressed as apparent affinity constants of MAbs OC125, OV-TL 3 and 139H2 for OVAR-3 cells were 1 x 10(9) M-1, 1 x 10(9) M-1, and 1 x 10(8) M-1, while the number of antigenic determinants were 5 x 10(6), 1 x 10(6) and 7 x 10(6), respectively. In OVCAR-3 bearing nude mice the blood half-lives of the MAbs as IgG and F(ab')2 were approximately 50 h and 6 h, respectively. Maximum tumour uptake for the whole MAbs OC125, OV-TL 3, 139H2 and a control MAb 2C7 was 8.5%, 17.7%, 11.1% and 2.5% of the injected dose g-1, reached at 72 h after injection. For the respective F(ab')2 fragments, the maximum values were 5.2%, 10.0%, 5.5% and 1.9% of the injected dose g-1, reached between 6 h and 15 h. Tumour to non-tumour ratios were more favourable for the F(ab')2 fragments as compared to those for MAbs as IgG. Biodistribution in mice bearing a control tumour confirmed the specificity of tumour localisation of MAbs OC125, OV-TL 3 and 139H2. After injection of a tracer dose of 10 microCi of radiolabelled MAbs OC125, OV-TL 3 and 139H2 as IgG, tumours received 38 cGy, and 9 cGy. In our OVCAR-3 model, a ranking in efficiency in tumour localisation would indicate MAb OV-TL 3 as most favourable MAb, but cross-reactivity with subpopulations of human white blood cells might hamper its clinical use. Dosimetric data indicate a 4-fold higher radiation absorbed dose to tumours for IgG compared with F(ab')2 fragments.

Effect of paclitaxel (Taxol) on CA 125 expression and release by ovarian cancer cell lines.

Two ovarian cancer cell lines, OVC432 and the newly established CVU4I, were used to study the effect of Taxal on cell growth and simultaneous CA 125 antigen expression. Growth of both cell lines was effectively inhibited by drug concentrations of 0.1 microM and higher. Complete inhibition of cell growth may result from high concentrations of Cremophor EL present in the Taxol formulation. Immunohistochemical analysis demonstrated that both cell lines retained the CA 125 expression on the cell surface during exposure to paclitaxel. This was reflected in a constant statistically significant correlation between cell numbers and CA 125 concentrations found in cell lysates. CA 125 levels in the culture medium showed a significant relation to cell numbers and, consequently, to the response of the cell line to the administered anticancer drug. It may be concluded from this study that CA 125 seems to be a reliable tumor marker in monitoring tumor response during paclitaxel treatment.

Escalating protein doses of chimeric monoclonal antibody MOv18 immunoglobulin G in ovarian carcinoma patients: a phase I study.

BACKGROUND: Successful first-line treatment of ovarian cancer does not incite cure. Recurrent disease often shows an increased resistance to chemotherapeutic agents. Therefore, other treatment modalities, like (radio)immunotherapy using tumor-associated monoclonal antibodies, should be considered. Chimeric MOv18 (c-MOv18) localizes well in ovarian carcinoma tissue. We investigated the safety of a single intravenous (i.v.) infusion of increasing doses of c-MOv18 in ovarian carcinoma patients. METHODS: Fifteen patients received c-MOv18 (from 5 mg to 75 mg). Safety was determined by recording vital signs; by hematological, biochemical, and urinary analyses; and by the human-antichimeric antibody (HACA) response. Five patients received c-MOv18 labeled with a tracer dose of iodine-131 to analyze the pharmacokinetics and biodistribution in blood, urine, and tissue biopsies at surgery. RESULTS: Administration of c-MOv18 IgG was uneventful. No significant changes in hematological, biochemical, or urine profiles were noted at any time postinjection (p.i). Starting with a dose of 50 mg, all patients experienced side effects, like fever, headache, and nausea/vomiting, maximally Grade II (World Health Organization toxicity scale). No HACA response was found up to 12 weeks p.i. The mean elimination half-life after infusion of 30-75 mg c-MOv18 was significantly higher compared with infusion of 1 mg. Absolute amount of c-MOv18 in carcinoma tissue increased with increasing c-MOv18 doses. CONCLUSIONS: Intravenous administration of c-MOv18 IgG in a dose up to 75 mg is safe, inducing only minor side effects at doses of 50 mg or higher. In view of the characteristics of c-MOv18, this antibody might be applicable as an unmodified antibody or as an immunoconjugate in the treatment of ovarian carcinomas.

Hexokinase isoenzymes from anaplastic and differentiated medullary thyroid carcinoma in the rat.

The activity, isoenzyme distribution and compartmentation of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were compared in slowly growing, well-differentiated medullary thyroid carcinoma (DMTC) and rapidly proliferating anaplastic thyroid carcinoma (AMTC) in the rat. Individual isoenzymes from either soluble or particulate fractions after solubilization were obtained by fast protein liquid chromatography and were kinetically analyzed either in soluble form or after (re)binding to rat liver mitochondria. These studies were undertaken to test the hypothesis that the growth rate of tumors is correlated with the activity of mitochondrial-bound hexokinase in our tumor system. In contradiction to this hypothesis, we found no difference in either enzyme activity or compartmentation of both kinds of tumors. The major part of enzyme activity was soluble (73 and 78% in DMTC and AMTC respectively). In addition, no major differences were observed in the kinetic properties of the individual isoenzymes of both tumors. Only soluble type II hexokinase from AMTC had a slightly decreased apparent Km for glucose. There appeared to be some differences in isoenzyme composition: both tumors contained type I and type II hexokinase in the soluble as well as in the particulate fractions. However, the proportion was shifted in favor of type II hexokinase in the soluble fraction of AMTC. Additional findings of this study were the following: the affinity of type II hexokinase to both substrates glucose and MgATP2- was significantly less compared to type I hexokinase. However, the inhibition constant for glucose-1,6-diphosphate of both isoenzymes was exactly the same. The bound form of both isoenzymes had the same substrate affinities as the soluble form but was considerably less inhibited by glucose-1,6-diphosphate. In the latter respect, type I and type II hexokinase behaved in the same way.

Tumor uptake of intravenously administered radiolabeled antibodies in ovarian carcinoma patients in relation to antigen expression and other tumor characteristics.

To study factors that possibly influence the heterogeneous tumor uptake of radiolabeled antibodies, tissues from 34 ovarian-carcinoma patients were obtained 2 to 8 days after i.v. injection with radiolabeled murine OV-TL3 or chimeric MOv18 (cMOv18). The tumor uptake and the ratio of tumor to normal tissue (T/NT) were studied in relation to the histopathological classification, prior treatment, site of tumor, time interval, antigen expression, volume percentage of (malignant) epithelium in the tumor tissue, and the size of the tumor. The results of immunoscintigraphy were also included. In addition, autoradiography using storage phosphor technology was performed on tissue sections from patients injected with iodine-labeled cMOv18. Tumor uptake varied largely, not only between patients, but also between tumor deposits within the same patient. Uptake of OV-TL3 F(ab')2 was higher than of cMOv18 F(ab')2, but the T/NT ratios were similar. The antibody uptake was positively correlated with the pattern of antigen expression and inversely correlated with the time interval between injection and surgery. No correlation was observed with any of the other factors studied. The visibility with immunoscintigraphy was related to the size of the detected lesion, but not to the other factors studied. Autoradiography showed that antibodies preferentially localized in areas with cancer cells, which were immunohistochemically positive for MOv18. In areas with weak antigen expression, autoradiography revealed less activity. The antigen expression by the tumor is an important factor for estimation of the tumor uptake of radiolabeled antibodies.