An experimental immunoscintigraphic study with anti-CEA monoclonal antibody (DG2).

Monoclonal antibody D11-DG2 (DG2) against carcinoembryonic antigen (CEA) was examined for suitability for radioimmunodetection of human tumors grown in nude mice. Antibodies DG2 and a control antibody of the same IgG1 subclass were labeled with 131I and injected into mice bearing one of three types of CEA-containing tumors (cell lines LS 174T, HT-29 and Rec S) and/or a CEA-negative tumor (Rec R). Gamma-camera imaging and distribution studies revealed that CEA-containing tumors selectively accumulate DG2 but Rec R does not. As the tumors differ in CEA-content, the highest accumulation of 131I-DG2 (corresponding to the best scintigraphic imaging) was found in LS 174T tumors, intermediate in Rec S and lowest in HT-29 tumors. The mean tumor-to-blood ratios on the sixth day after antibody administration were 4.6, 3.2, and 2.1, respectively, in the control experiments the value of this parameter was always lower than 1. The results showed the applicability of DG2 for immunoscintigraphic studies in patients. Furthermore, a positive correlation was found between the uptake of anti-CEA antibody and CEA-content in the tumors.

Distribution, kinetics and immunoscintigraphy of 131-I labelled intact antifibrinogen-fibrin antibody (AbFbg) and its F(ab')2 fragment.

131-I-labelled anti fibrin-fibrinogen antibody (AbFbg) was compared with its F(ab')2 fragment in distribution studies and by immunoscintigraphy with a view to tumour visualization in tumour bearing rats. The distribution studies indicated that the intact antibody is more concentrated in tumour tissue than the F(ab')2 fragment. By 168h after injection, when tumour-to-tissue ratios were highest in the majority of tissues, the tumour concentration of intact antibody was 3 to 4 times that of the F(ab')2 fragment. The intact antibody is more suitable than the F(ab')2 fragment for tumour imaging especially in the abdominal region where the highest tumour-to tissue ratios were obtained with intact antibody in liver, spleen, intestines and kidneys.

[Immunoscintigraphy in experiments and clinical practice: its possibilities, advantages and limitations]. / Imunoscintigrafie v experimentu, klinice, její moznosti, výhody a omezení.

The authors present their experience gained in preparing, isolating and labeling antibodies with radionuclides for the purpose of using them in immunoscintigraphy. The experimental part includes results obtained with different labeled antibodies and their F/ab/2 fragments in distribution studies, involving also immunoscintigraphic imaging of tumors. The clinical part presents results of immunoscintigraphy obtained with the commercial antibody kits Iodomab and Imacis in patients with tumors of the digestive tract.

Antiferritin antibodies in immunoscintigraphic detection of human tumor xenografts.

Affinity-purified antibodies against human placental ferritin and their F(ab)2 fragments labeled with 131I were examined for suitability for radioimmunodetection of ferritin-containing tumors. The nude mouse model (BALB/c, nu/nu) with xenografts of HeLa cell tumors and human adenocarcinoma of the rectum (with proven ferritin content) was used. Gamma-camera imaging and tissue distribution studies revealed that both kinds of tumor selectively accumulate antiferritin antibodies and their fragments. In large necrotic tumors nonspecific uptake of radiolabeled normal IgG occurred, but otherwise there was no tumor localisation. This study, in accordance with the literature, confirms the utility of antiferritin antibodies for the detection of human tumors in an animal model.

Ferritin-bearing lymphocytes in Hodgkin's disease.

In view of the reported association of Hodgkin's disease (HD) and ferritin, ferritin-bearing lymphocytes were followed during 2-year period in 79 HD patients. Indirect immunofluorescent method was used to evaluate the percentage of ferritin positive cells. In 22 untreated patients a high percentage of ferritin-bearing circulating lymphocytes (mean value 37.3%) was found. In regard to the extent of the disease higher values were found in clinical stage III and IV (mean value 40.6%) as compared to the stage I and II (mean value 26.2%). Similarly, 17 patients in relapse and with disease progression had mean values 41%. These proportions of cells were significantly lower in 44 patients in complete remission with mean value of 8.7% (60 examinations). In 30 healthy controls the mean value was 1.4%. Repeatedly performed examinations of ferritin-bearing lymphocytes during the follow-up period in 17 patients showed to be an important prognostic tool. A negative correlation of ferritin-bearing lymphocytes with E-rosette-forming cells was found. Iron content in peripheral blood lymphocytes was confirmed cytochemically after pre-incubation with antiferritin antibody. The results support the presumed role of ferritin in impaired cellular immunity in HD and suggest diagnostic and prognostic value of the examination of ferritin-bearing lymphocytes in HD.

Identification of leukemia cell-derived inhibitory activity (LIA) in conditioned media from human myeloid leukemic cell line ML-2.

Conditioned media from the human myeloid leukemic cell line ML-2 contain a factor that inhibits the entry of normal CFU-GM into S phase of mitotic cycle as measured by the 3H-TdR suicide technique. This factor was detected in conditioned media prepared by incubating 5 X 10(6) ML-2 cells/ml or 1 X 10(6) ML-2 cells/ml in serum-free RPMI for 5 or 24 hours respectively, and was isolated by ultrafiltration through an XM 300 Diaflo membrane followed by chromatography on Sepharose 6 B. Ferritin, prepared from human placenta, had the same inhibitory effect on CFU-GM. Antibodies against human placental ferritin completely inactivated the inhibitory effect of both human placental ferritin and the factor released from ML-2 cells. The inhibitory activity produced by the cell-line ML-2 was considered as LIA (leukemia cell-derived inhibitory activity) earlier found in HL-60 cell line and AML and CML cells.

Monoclonal antibodies against human urinary bladder carcinomas: selectivity and utilization for gamma scintigraphy.

Mouse monoclonal antibodies to human urinary bladder carcinoma cells have been examined by indirect membrane immunofluorescence using a panel of 27 human cell lines. Two of the monoclonal antibodies, 7E9 (IgG3) and S2C6 (IgGl), were found to distinguish between urinary bladder carcinoma cells and normal urothelium. The third monoclonal antibody, T24.06.5(IgGl), discriminated among cell lines of urothelial and non-urothelial origin but did not distinguish between urinary bladder carcinoma and normal urothelial cells. None of the of the antibodies was found to be strictly selective, and occasional cross-reactions with unrelated cell types were observed. The monoclonal antibody 7E9, showing the highest degree of selectivity, was further examined by an indirect immunoperoxidase technique on frozen tissue sections from 19 patients. The antibody reacted with all (7/7) bladder carcinomas examined and gave negative results with control normal bladder mucosa (0/8) and unrelated tumor tissue (0/4) sections. The 7E9 antibody was purified by protein A affinity chromatography, labeled with 131I and used for gamma-scintigraphy in nude mice xenografted with human urinary bladder carcinoma T24. The 7E9 antibody was capable of locating the T24 xenografts in nude mice; it localized preferentially in the T24 tissue compared to normal mouse tissues. The T24 xenografts could not be detected by gamma-scintigraphy with 131I-labeled monoclonal antibody against human mammary carcinoma cells and two other control antibodies. Likewise the 131I-labeled 7E9 antibody was not capable of locating human mammary carcinoma xenografts in nude mice.

Tumour localization of radiolabelled monoclonal antibody in mice bearing human urinary bladder (T24) carcinoma xenografts.

Selectivity of mouse monoclonal antibody 7E9 (IGG3) directed against human urinary bladder carcinoma cells has been examined by indirect membrane immunofluorescence, using a panel of 31 human cell lines. The 7E9 monoclonal antibody discriminated between urinary bladder carcinoma cells and normal urothelium or cells of non-urothelial origin, although occasional reactions with bladder carcinoma-unrelated cell types were observed. The 7E9 antibody was purified by protein A affinity chromatography, labeled with 131I and used for gamma scintigraphy in nude mice xenografted with human urinary bladder carcinoma T24. The 7E9 antibody was capable of locating the T24 xenografts in nude mice; it localized preferentially in the T24 tissue compared to normal mouse tissues. The T24 xenografts could not be detected by gamma scintigraphy with 131I-labelled monoclonal antibody against human mammary carcinoma cells and two other control antibodies. Likewise, the 131I-labelled 7E9 antibody was not capable of locating human mammary carcinoma xenografts in nude mice.

Immunoscintigraphy and distribution study of experimental tumours using 131I-labelled anti-fibrinogen antibodies.

131I-labelled antibodies to the fragment E of human fibrin, anti-rat fibrinogen, non-immune rabbit IgG and 67Ga-citrate were used for scintigraphy and distribution studies in experimental tumours in rats. The best visualisation was achieved with 131I-labelled anti-rat fibrinogen antibodies at intervals longer than 48 h after administration. Tumour to tissue ratios found in distribution studies performed formed more than 48 h after administration were higher in most examined tissues when using 131I-labelled antibodies than those obtained with 67Ga-citrate.

Lymphocyte nucleoli activation and changes of the mononuclear phagocytic system in mice during 3-methylcholanthrene carcinogenesis.

After an intra-muscular application of 3-methylcholanthrene (MCA) in a dose of 0.2 mg/animal to SPF mice, strain C57B1/10, changes in the lymphocyte nucleoli activation (LNA) and changes of the mononuclear phagocytic system (MPS) in blood and tissues were monitored. The tests were carried out after the first and sixth weeks of the MCA administration, when the tumours were not yet palpable, and 12 weeks after the MCA injection, when the tumours were manifest in 100% of animals. In the first and sixth week after MCA application, the number of non-activable lymphocytes with micronucleoli increased at the expense of the resting ring-shaped forms. In the 12th week, a highly significant drop of active lymphocytes with nucleolonemas and compact nucleoli and particularly a high number of afunctional lymphocytes with micronucleoli were observed in tumour-bearing mice. The phagocytic activity of peripheral mononuclears was significantly increased in the same time interval. MPS activity, monitored on the basis of the colloidal carbon clearance, was stepwise inhibited in animals with originating tumours. It should be pointed out that the LNA test and probably the changes in carbon clearance were early indicators of involvement in chemical carcinogenesis.

Ceruloplasmin in Hodgkin's disease.

In 56 patients with Hodgkin's disease different clinical and histological stages the activity of polyphenoloxidase was repeatedly examined in the course of the disease as an indicator of serum ceruloplasmin concentration. In untreated patients the ceruloplasmin concentration was on average twice as high as in healthy controls. Repeated examinations made in the course of the disease demonstrated a good correlation of the values with the clinical condition of the patient, with the exception of some terminal stages and conditions following repeated chemotherapy, when liver damage was expected. The decrease to control levels in the course of therapy indicated the onset of remission. In contrast, increased levels indicated progression of the disease and necessity of treatment prolongation. Excluding intercurrent infections, higher ceruloplasmin levels above those found at remission were a clear sign of the progression. The correlation of ceruloplasmin values and erythrocyte sedimentation rate (1 h) was highly significant. The diagnostic and prognostic value of ceruloplasmin and/or serum copper determination is discussed.

Immunoglobin levels in patients with Hodgkin's disease.

The authors examined a course of immunoglobulin values (IgA, IgG, IgM) in 40 patients with the Hodgkin's disease and repeatedly in 11 healthy persons. In the patients they followed the levels of the immunoglobulins during the disease, up to 5 years. In untreated patients there were statistically significant increased IgA levels and insignificant increase of IgG. In the course of remission the immunoglobulin values were normalized. The radiotherapy did not affect significantly the value of immunoglobulins, the combined chemotherapy (COPP) led to a drop of this level. The levels of immunoglobulins are recovered after chemotherapy in the case of successful treatment. In terms of the prognosis the authors consider as unfavorable high increase in the immunoglobulin levels in untreated patients and their rapid drop during the chemotherapy. They observed an average preterminal drop of the IgA by 27%, IgG by 23% and IgM by 44% with respect to controls.

Isolation of alpha-1 fetoprotein.

A suitable method of the isolation of alpha-1 fetoprotein for the needs of enzyme immunoassay of this oncofetal antigen is described. By combining isoelectric focusing and "indirect" affinity chromatography the preparation of alpha-1 fetoprotein was obtained that was not contaminated with IgG, contrary to the isolation performed by means of "direct" affinity chromatography on a carrier with coupled anti-alpha-1 fetoprotein antibodies, or other immunochemical methods that usually yielded contaminated preparations. Neither disc electrophoresis in PAA gel, immunoelectrophoresis, double radial immunodiffusion, nor biological experiments revealed any traces of ballast proteins in the resulting preparation; it seems suitable both for the preparation of monovalent antisera of a sufficient avidity, and as a standard for enzyme immunoassay.