Keigo Endo

The design of a pentavalent 99mTc− dimercaptosuccinate complex as a tumor imaging agent

In our search for a technetium-tumor seeking agent, the chemical characteristic of polynuclear Ga-citrate attracted our attention. On this basis, we selected the ligand dimercaptosuccinic acid (DMS) and appropriate labeling conditions in the design of 99mTc(V)-DMS. Chemical characterization was performed by thin layer chromatography, electrophoresis, Sephadex column chromatography and spectrophotometric studies at the chemical concentration (99Tc). Biodistribution in mice bearing Ehrlich ascites tumor and scintigraphic images in VX-2 tumor-bearing rabbit, indicated the great applicability of 99mTc(V)-DMS as a new tumor imaging agent. Its distinctive characteristic, different from the kidney imaging agent (99mTc-DMSA) is demonstrated and the biological implication of hydrolytic polynucleation of pentavalent technetium, through an anionic specie Tc(V)O3-(4), in the tumor cell is discussed.

Normal bronchial mucus contains high levels of cancer-associated antigens, CA125, CA19-9, and carcinoembryonic antigen

The presence of cancer‐associated antigens CA125, CA19‐9, and carcinoembryonic antigen (CEA) in apparently normal respiratory system was demonstrated histochemically and immunochemically. Epithelial cells lining central airways (trachea, bronchi, and bronchioli) and respiratory glands were specifically stained by antibodies recognizing CA125, CA19‐9, and CEA. Most, if not all, bronchial mucus obtained from patients without pulmonary diseases during general anesthesia contained remarkably high levels of CA125, CA19‐9, and CEA ranging from 190 to 41,000 U/ml (594–4803 U/mg protein), 210 to 95,000 U/ml (294–197,917 U/mg protein), and 6 to 940 ng/ml (14–209 ng/mg protein), respectively, whereas serum antigen levels were normal in all cases examined. These results suggest that CA125, CA19‐9, and CEA are synthesized and secreted by normal epithelial cells of central airways and/or respiratory glands and that these substances are not specific indicators of abnormal cellular activity.

Serum CA 19-9 concentrations and computed tomography findings in patients with pancreatic carcinoma.

Carbohydrate antigen (CA) 19‐9 is a new tumor markerdefined by a monoclonal antibody. Serum CA 19‐9 concentrations and computed tomography (CT) findings were studied in 55 patients with histologically proven adenocarcinomaand in 22 patients with chronic pancreatitis. CA 19‐9 was useful in 83% of cases for the differential diagnosis between pancreatic carcinoma and chronic pancreatitisand serum CA 19‐9 levels in pancreatic carcinoma were highly related to the size of tumors. Serum CA 19‐9 levels greater than 37 U/ml were seen in patients with a tumor of less than 3 cm3 to 5 cmand greater than 5 cm in diameter 13% (1/8)90% (19/21)and 92% (24/26) of casesrespectively. Tumor locationhoweverwas unrelated to serum CA 19‐9 value. These results indicated that the measurement of serum CA 19‐9 concentrations would be useful in mostif not allcases for the differential diagnosis between pancreatic carcinoma and chronic pancreatitisand for the evaluation of tumor burden in patients with pancreatic carcinoma.

Small carcinoma of the pancreas. Clinical and pathologic evaluation of 17 patients.

The clinical and pathologic characteristics of 17 small carcinomas (less than 2 cm in diameter) of the pancreas are reviewed in this article. All the tumors were located in the head of the pancreas, and the clue to the diagnosis was jaundice in ten patients and abdominal pain in seven. Carcinoembryonic antigen (CEA) and CA 19‐9 were not reliable markers for detecting small carcinomas of the pancreas. Ultrasonography (US), computerized tomography (CT), percutaneous transhepatic cholangiography (PTC), and endoscopic retrograde cholangiopancreatography (ERCP) were useful diagnostic tools. Lymph node metastases were found in 41% of affected patients, capsular invasion in 24%, retroperitoneal invasion in 24%, and portal system involvement in 29%. In five patients the carcinoma was Stage I; in eight patients, Stage II; in two patients, Stage III, and in two patients, Stage IV. Fifteen patients with Stages I to III and one patient with Stage IV underwent curative pancreaticoduodenectomy or total pancreatectomy, and one patient with liver metastasis and Stage IV underwent noncurative pancreaticoduodenectomy. The cumulative 4‐year survival rate was 37%. Although four patients with Stage I disease lived for more than 48 months, the survival period of the 12 patients with Stages II to IV disease was less than 25 months. Thus, small carcinoma of the pancreas is not always curable; however, a small, localized lesion without any extratumoral extension can be resected with a chance of cure.

Cystic papillary carcinoma of the thyroid gland: A new sonographic sign

Cystic thyroid lesions are generally considered to be benign and managed by repeated aspiration and/or biopsy. We report characteristic sonographic signs in eight cases of cystic papillary carcinomas of the thyroid gland. In all eight cases, ultrasonography (US) revealed mostly cystic lesions with solid excrescences protruding into the cyst. These nodules contained multiple punctate echogenic foci suggesting calcification. In three of these cases the initial fine needle aspiration biopsy was negative. The calcified nodule in cyst sign was found to be very specific for cystic thyroid carcinoma in a review of the ultrasound findings in 115 patients with nodular thyroid lesions. Careful evaluation and/or surgery should be recommended in patients who have such characteristic sonographic findings even if the fine needle aspiration result is negative.

Myocardial uptake of antimyosin monoclonal antibody in a murine model of viral myocarditis.

The myocardial uptake of 125I- and 131I-antimyosin monoclonal antibody Fab in experimental myocarditis in BALB/c mice induced by encephalomyocarditis virus was studied. The biodistribution of 125I-antimyosin demonstrated that the highest ratio of radioactivity appears in the heart of infected mice on day 14 (the ratio of percent dose per gram for the organ to percent dose per milliliter for blood; 9.75 +/- 2.79 vs. 1.27 +/- 0.78 at 24 hours in inoculated mice vs. control mice). There was no statistically significant difference between the mean activity ratios of tissues other than the heart in control and inoculated mice. The uptake ratio for the heart increased significantly 3 days after virus inoculation and reached a maximum on day 14 when myocardial lesions were most extensive and prominent. The uptake ratio decreased significantly, but it still remained high compared with controls on day 28 when cellular infiltration had decreased and fibrosis was evident. The scintigraphic images obtained with 131I-antimyosin monoclonal antibody clearly demonstrated that visualization of the heart in experimental myocarditis was possible 24 hours after administration of radiotracer, and localized activity was still observed in the 48-hour image. We conclude that antimyosin monoclonal antibodies localize selectively in the heart from the acute to subacute stage of viral myocarditis. These findings indicate that antimyosin scintigraphy is a reliable noninvasive method for the evaluation of patients suspected of having myocarditis.

Human/mouse chimeric antibodies show low reactivity with human anti-murine antibodies (HAMA)

Human anti-murine antibody (HAMA) response is a serious problem in the repeated infusion of murine monoclonal antibodies (MoAbs). HAMA positive sera were obtained from seven patients with colorectal cancer, pancreas cancer, malignant melanoma or myocardial infarction who had previously received radiolabelled MoAbs. The nature of HAMA was analysed using size exclusion high performance liquid chromatography (HPLC) after incubating with radiolabelled MoAbs including IgG, Fab or human/mouse chimeric Abs. Immune complexes composed of HAMA and MoAbs were formed. The percentage of radioactivity with a high molecular weight was related to HAMA levels determined by enzyme linked immunosorbent assay. Most radioactivity present in immune complex shifted to the antibody fraction after the addition of normal murine serum. All of seven sera were reactive with all four murine IgGs and this suggests that HAMA in these patients recognised the constant region of MoAbs. In one patient, HAMA was considered to recognise the variable region and to be anti-idiotypic. There was no significant binding with human/mouse chimeric Abs in any HAMA positive serum, although five out of seven patients were reactive with murine MoAb Fab, indicating that HAMA was composed of Abs responsive to the CH1 or CL region of murine IgG. These results suggest that (1) HAMA was composed of Ab responsive to Fc portion and/or CH1 or CL region of murine IgG, and (2) human/mouse chimeric Abs look promising in the repeated infusion of MoAb in HAMA positive patients.

Analysis of the levels of CA125, carcinoembryonic antigen, and CA19-9 in the cervical mucus for a detection of cervical adenocarcinoma

To verify whether analysis of the levels of CA125, carcinoembryonic antigen (CEA), and CA19‐9 in the cervical mucus is effective for a detection of cervical adenocarcinomas or not, simultaneous measurement of these three tumor markers in the cervical mucus samples from women without gynecologic disorders, with leiomyoma, with cervical squamous cell carcinomas, and with cervical adenocarcinomas was performed. Extremely high levels of CA125 with low levels of both CEA and CA19‐9 were demonstrated in the cervical mucus samples from women without gynecologic disorders and with leiomyoma. The cervical mucus samples from cervical adenocarcinomas showed low CA125 levels with extremely high CEA and/or CA19‐9 levels. Therefore, analysis of the levels of these three tumor markers in the cervical mucus possibly helps in the diagnosis of cervical adenocarcinomas if CEA and/or CA19‐9 show extremely high levels. When a ratio of (CEA + CA19–9)/CA125 was calculated, all women without gynecologic disorders and with leiomyoma showed a ratio <0.5, whereas ten of 11 cases of cervical adenocarcinomas had a ratio ≥0.5. Only one case of microinvasive adenocarcinoma showed a ratio <0.5. Accordingly, the ratio ≥0.5 strongly suggested an existence of cervical adenocarcinoma, although it included some cases of squamous cell carcinomas (four of 17 cases).

STUDIES ON THYROTROPHIN RECEPTOR ANTIBODIES IN PATIENTS WITH EUTHYROID GRAVES' DISEASE

Thyroid stimulating antibodies (TSAb) and TSH‐binding inhibitor immunoglobulins (TBII) were assessed in 30 patients with euthyroid Graves disease. TSAb were detected in 24 cases (80.0%), the incidence being not significantly different from that in hyperthyroid Graves disease (29/30,97.6%). On the other hand, the incidence of TBII in patients with euthyroid Graves disease (12/30, 40.0%) was significantly lower than that in patients with hyperthyroid Graves disease (30/30, 100.0%). The mean TSAb and TBII activities in the euthyroid patients were significantly lower than in the hyperthyroid patients (P < 0.f005 and P < 0.001, respectively). Both TBII and, more closely, TSAb activities correlated with T3‐nonsuppressibility and inhibition of serum TSH response to TRH stimulation. The findings supported the stimulation in vivo of the thyroid by these antibodies.

Preparation of 67Ga-labeled antibodies using deferoxamine as a bifunctional chelate an improved method

Radionuclides or anti-cancer drugs may be coupled to antibodies for specific transport to target tissues. We have previously reported that several proteins could be rapidly and efficiently labeled with gallium (67Ga) by using deferoxamine (DFO) as a bifunctional chelating agent. In the present paper, we have described the use of hetero-bifunctional agents for the conjugation of DFO with antibodies and investigated the effect of coupling agents on in vitro properties and biodistribution of 67Ga-labeled antibodies. 67Ga-labeled monoclonal antibodies retained antigen-binding activity when prepared under optimum conditions. The use of hetero-bifunctional reagents, such as succinimidyl 6-maleimido-hexanoate (EMCS) or N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), which link thioether bonds and disulfide bridges prevented the formation of polymerized antibodies. Although high non-specific uptake in the liver was observed with radiolabels prepared by the homo-bifunctional agent glutaraldehyde, uptake in the liver was low with conjugates linked by hetero-bifunctional agents. 67Ga-labeled antibodies with thioether bonds showed in vivo stability, but the clearance from the circulation was the fastest with the radiolabel holding disulfide bonds. The coupling reagents used to link DFO and antibodies greatly influenced both in vitro properties and in vivo distribution of labeled antibodies and 67Ga-labeled antibodies provide a good model for the study of coupling methods and biodistribution of antibody conjugates.