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.     

Biodistribution of iodine-125 and indium-111 labeled OV-TL 3 intact antibodies and F(ab')2 fragments in tumor-bearing athymic mice.

The monoclonal antibody OV-TL 3, directed against an ovarian carcinoma-associated antigenic determinant, was tested as a vehicle for radioimmunolocalization of ovarian carcinomas in athymic mice bearing NIH:OVCAR-3 xenografts. The biodistribution of intact. OV-TL 3 was compared with the distribution of OC 125. Tumor uptake with OV-TL 3 was significantly higher than with OC 125, and almost 7 times higher than with a non-specific control antibody (OV-TL 19). Administration of a mixture of intact OV-TL 3 and OC 125 did not improve tumor uptake in comparison with OV-TL 3 alone. Subsequently, intact OV-TL 3 and its F(ab')2 fragments were labeled with either 111In or 125I. The highest tumor uptake was obtained with 111In-labeled intact OV-TL 3 (14.7% ID/g, 48 hr p.i.). For both antibody forms uptake of 111In in liver, spleen and kidneys was very high. Furthermore, 111In cleared more slowly from most tissues than 125I. As a result, tumor/tissue ratios with 111In-labeled OV-TL 3 were lower than with 125I-labeled OV-TL 3. The highest tumor/tissue ratios (6.9 to 53) were obtained with 125I-labeled OV-TL 3 F(ab')2 fragments, 48 hr post injection. 111In-labeled OV-TL 3 F(ab')2 has already been shown to be a clinically useful label for the detection of ovarian cancer. The results of our comparative animal study suggest that these clinical results may even be improved by using 123I-labeled OV-TL 3 F(ab')2.     

Biodistribution of indium-111-labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas

The biodistribution of 111In-labeled monoclonal antibody (MAb) OC 125 was studied after i.p. injection in 28 patients who underwent surgery for ovarian carcinoma. Group I (eight patients) received intact 111In-labeled OC 125 MAb, Group II (three patients) intact 111In-labeled irrelevant NS, Group III (five patients) intact 111In-labeled OC 125 MAb associated with 20 mg of the same unlabeled MAb and Group IV (12 patients) F(ab')2 fragments of 111In-labeled OC 125 MAb. The patients were operated on 1 to 3 days after i.p. injection, and the surgeon removed large tumor fragments and/or small tumor nodules and, in some patients, collected the residual perfusion fluid from which malignant cell clusters were isolated. Uptake by large tumor fragments at 24 h was low: 0.0031 +/- 0.0032% injected dose per gram (%ID/g) for Group I and 0.0024 +/- 0.0022%ID/g for Group IV. It was moderately higher than that of Group II (0.0014 +/- 0.0006%ID/g) and Group III (0.0015 +/- 0.0007%ID/g). Uptake by small tumor nodules (0.1302 +/- 0.0802%ID/g at 72 h for Group I) and malignant cell clusters (median: 0.3322, with a maximum value of 4.1614%ID/g at 24 h for Group IV) was markedly higher. Tumor-to-normal tissue ratios with OC 125 MAb [intact or F(ab')2 fragments] ranged between 0.1 and 8.5 for large tumor fragments and 2 and 8,700 for small tumor nodules and malignant cell clusters. It would thus appear that RIT is feasible if an appropriate radionuclide can be selected for antibody labeling.     

Marker profile of mesothelial cells versus ovarian carcinoma cells

We investigated the marker profile of human ascitic and cultured mesothelial cells, and compared it to that of ovarian carcinoma cells which are related in terms of their histogenesis, unrelated colon carcinomas being used as reference. Mesothelial and ovarian carcinoma cells could not be distinguished by (intermediate) filament typing, using monoclonal antibodies (MAbs) to keratins, vimentins and desmins. Colon carcinomas differed from mesothelial cells and ovarian carcinomas by the absence of keratin-7 filaments. The epithelial marker BW 495/36 was completely negative on mesothelial cells and positive on all ovarian and colon carcinoma cells. While CEA was found on about 85% of all colon carcinomas, CEA expression on mesothelial cells and ovarian carcinoma cells was below 20%. The ovarian carcinoma markers (OV-TL 3, OV-TL 10, OC 125, MOV 18) were strongly positive on ovarian carcinomas and negative on colon carcinomas (or limited to traces of immunofluorescence on some samples). Although the mesothelial cells showed weak or negative reactivity with these markers, OC 125 antigen was found by immunoelectron microscopy on the surface of cultured mesothelial cells, and was shed in the culture supernatant at concentrations of 50, 28, and 25 CA 125 U/ml/10(4) positive cells. This suggests that mesothelial cells may be responsible for the synthesis of CA 125 in ascitic fluid. The data indicate that ovarian carcinomas, mesothelial cells and colon carcinomas can be distinguished using a combination of anti-keratin antibodies with BW 495/36 and anti-ovarian carcinoma markers.     

Monoclonal antibodies that discriminate between human ovarian carcinomas and benign ovarian tumours

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against ovarian carcinomas were obtained after immunizing mice with an undifferentiated human ovarian cystaden-ocarcinoma extract. The hybridoma cell supernatants were initially screened for a positive immunohistochemical reaction with ovarian carcinomas concomitant with a negative reactivity with a benign ovarian cystadenoma.The antibodies OV-TL 15 (IgM), OV-TL 30 (IgG1) and OV-TL 31 (IgM) reacted positively with 84, 84 and 74% of the ovarian carcinoma samples (n = 76) respectively. Reactivity with ovarian cysts and cystadenomas (n = 21), with non-ovarian carcinomas (n = 77) and with normal human tissue samples (n = 63) was absent or limited to weak reactivity on incidental samples.All three antibodies were shown by immunoelectron microscopy to react with surface antigens (OA 15, OA 30 and OA 31) of ovarian carcinoma cells. Cross reactivity between OV-TL 15, OV-TL 30, OV-TL 31 and OC 125 monoclonal antibodies was excluded by competitive binding assays on NIH:OVCAR-3 cells.Antigen levels (OA 15 and OA 31) in tumour extracts and cyst fluids were quantified by immunoradiometric assays and compared to the CA 125 antigen levels. High levels of CA 125, OA 15 and OA 31 were found in cyst fluids from ovarian cancers. In benign ovarian cyst fluids, however, the CA 125 content was also high while OA 15 antigen was hardly detectable. The OA 31 antigen was present at relatively low levels.The IRMA data confirmed the immunohistochemical data showing that, in contrast to OC 125, the newly developed MAbs OV-TL 15, OV-TL 30 and OV-TL 31 discriminate between benign ovarian cystadenomas and malignant ovarian cancers.     

ETN-1: a new human endometrial carcinoma cell line producing ascites and distant metastases in nude mice

A human carcinoma cell line (ETN-1) has been established from a skin metastasis of a moderately differentiated adenocarcinoma of endometrial origin. The cell line has been so far maintained for 27 months through 55 passages, growing as a monolayer as well as in 3-dimensional clusters with a population doubling time of 72 hr. The number of chromosomes per cell varied from 39 to 107 (average number 61.0 +/- 19.8), with a modal number of 46-48. Seven clonal marker chromosomes were detected. Flow cytometric analysis revealed a population of pseudo-tetraploid cells (DNA index 2.1) next to a pseudo-diploid population (DNA index 1.1). The epithelial character of the cells was confirmed by a positive immunocytochemical reaction using monoclonal antibodies (MAbs) to different keratins, the epithelial cell markers BW 495/36 and HMFG-2, as well as by the presence of many junctional complexes. The tumour cells retained a positive reaction with the anti-ovarian carcinoma OV-TL 3, OV-TL 10 and OC 125 MAbs, although the reaction was markedly diminished in comparison with the original tumour. Tumour cells inoculated subcutaneously in nude mice produced well differentiated adenomatous tumour nodules with formation of glandular lumina and basal lamina. Tumour cells injected intraperitoneally produced malignant ascites and regional as well as distant metastases of adenomatous character.     

Biodistribution of indium-111-labeled OC 125 monoclonal antibody after intraperitoneal injection in nude mice intraperitoneally grafted with ovarian carcinoma

The purpose of this work was to study the biodistribution of 111In-labeled OC 125 monoclonal antibody (MAb) with known affinity for ovarian carcinomas in a nude mouse model grafted i.p. with a human ovarian cancer (NIH:OVCAR-3). Tumor uptake 24 h after i.p. injection was higher with intact 111In-labeled OC 125 MAb (28 +/- 7.44%ID/g) than with 111In-nonspecific immunoglobulin (6.86 +/- 1.35%ID/g). The kinetics of tumor uptake also differed, showing a plateau followed by a drop at Day 7 with 111In-OC 125 MAb and a decrease beginning at 24 h with 111In-nonspecific immunoglobulin. Tumor-to-normal tissue ratios ranged between 29.91 +/- 11.85 and 0.68 +/- 0.15 with 111In-OC 125 MAb and between 4.50 +/- 1.06 and 0.53 +/- 0.04 with 111In-nonspecific immunoglobulin according to the normal tissues and the time points considered. Tumor uptake 2 h after injection was the same for F(ab')2 fragments as for intact MAb, whereas maximum uptake at 24 h (18.76 +/- 4.62%ID/g) was lower and was followed by a decrease at Day 4. Tumor-to-normal tissue ratios were in the same range, except for the tumor to blood ratio which was higher and the tumor to kidney ratio which was lower at 24 and 96 h. Maximum tumor uptake was higher after i.p. (30.77 +/- 4.76%ID/g) than i.v. (14.59 +/- 2.70%ID/g) injection. Instead of attaining the plateau noted after i.p. injection, tumor uptake after i.v. injection remained low at 2 h (2.11 +/- 1.66%ID/g), reaching its peak only after 96 h. 131I-OC 125 injected i.p., which reached maximum tumor uptake at 2 h (13.53 +/- 4.25%ID/g), showed tumor-to-tissue ratios ranging between 15.98 +/- 2.63 and 0.96 +/- 0.86, i.e., not very different from those with 111In. After i.p. injection of a radiolabeled colloid solution, maximum tumor uptake was reached at 96 h (20.22 +/- 5.35%ID/g), but with very high nonspecific uptake in liver (31.06 +/- 6.22%ID/g) and spleen (55.23 +/- 14.11%ID/g). These results indicate high, selective tumor uptake of 111In-OC 125 after i.p. injection and demonstrate the feasibility of i.p. radioimmunotherapy of ovarian carcinomas.     

Antibody-antigen complex formation following injection of OC125 monoclonal antibody in patients with ovarian cancer

Monoclonal antibody (MAb) OC125 binds to approximately 80% of epithelial ovarian cancers. Serum antigen, CA125, can be detected in these patients. 131I-OC125-F(ab')2 was injected into 5 ovarian carcinoma patients with preinjection serum levels of 150 to 9,000 CA125 U/ml. Patients received the antibody intravenously in doses ranging from 0.46 to 0.94 mg with a specific activity of approximately 2.5 mCi/mg 131I. The half-life in the circulation was approximately 30 hr and was independent of serum CA125 levels. Clearance of 131I from the circulation fitted an open, one-compartment mathematical model. Gel filtration chromatography revealed antibody-antigen complexes in sera 15 min after injection of the radiolabelled antibody. By 5 days after injection, the free form of OC125 antibody could not be detected in the serum. The rate of complex formation correlated well with the observed preinjection serum CA125 levels. This direct correlation was verified in vitro using purified CA125 antigen and radiolabelled OC125 F(ab')2 fragments. The specific effects of complex formation on tumor localization remains unclear. However, the presence of complexes should not be ignored, when planning for diagnostic imaging or immunotherapy with OC125 or other MAbs reacting with circulating antigen.     

In vivo targeting of OV-TL 3 immunoliposomes to ascitic ovarian carcinoma cells (OVCAR-3) in athymic nude mice.

Specific binding of immunoliposomes to target tumor cells was investigated in a xenograft model (athymic nude mice) of i.p. growing human ovarian carcinoma (OVCAR-3). For the first time, quantitative evidence is presented that attachment of a tumor-specific antibody (OV-TL 3) dramatically enhances the association of liposomes with i.p. growing OVCAR-3 cells. The OV-TL 3-mediated binding of liposomes to the OVCAR-3 cells was rapid; 30 min after i.p. injection approximately 70% of the injected dose of OV-TL 3 immunoliposomes was associated with the OVCAR-3 cells while for unconjugated liposomes a value of only approximately 3% was obtained. At 2 h after injection, a maximal binding level of 84% was achieved in case of the OV-TL 3 immunoliposomes whereas the binding level of unconjugated liposomes was still about 3%. Twenty-four h after injection about 83% of the injected dose OV-TL 3 immunoliposomes still was associated with the OVCAR-3 cells, compared to about 10% of the injected dose of unconjugated liposomes. Accordingly, unconjugated liposomes disappeared from the peritoneal cavity much faster than the OV-TL 3 immunoliposomes. By comparison with immunoliposomes bearing irrelevant antibody, the specificity of the binding of the OV-TL 3 immunoliposomes to the OVCAR-3 cells was demonstrated. In addition, it was observed that the sustained high OV-TL 3 immunoliposome levels found in the peritoneal cavity are the result of both reduced particle clearance from the peritoneal cavity and the tenacious binding of the immunoliposomes to the tumor cells. Finally, data are presented showing that the degree of binding of OV-TL 3 immunoliposomes to OVCAR-3 cells in vitro and in vivo correlates positively with the antibody (Fab') density on the liposomes.     

Bikunin plus paclitaxel markedly reduces tumor burden and ascites in mouse model of ovarian cancer

Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression.     

Localization of the monoclonal antibody HMFG2 after intravenous and intraperitoneal injection into nude mice bearing subcutaneous and intraperitoneal human ovarian cancer xenografts

Xenografts (s.c. and i.p.) of human ovarian cancer, shown to express the tumor associated antigen defined by the monoclonal antibody HMFG2, were used to investigate in vivo localization of the radioiodinated antibody after i.p. and i.v. injection. Following i.v. injection, maximum uptake (31.4 +/- 3.5%/g s.c. tumor) was seen in s.c. tumors at 48 h after injection. Tumor:normal tissue ratios increased with time to 240 h. Uptake by ascites and i.p. tumors was less, with a maximum of 10.0 +/- 8%/g ascites reached at 16 h and 11.4 +/- 3.2%/g i.p. tumor at 96 h. After i.p. injection uptake in s.c. xenografts was maximal (14.8 +/- 2.0%/g) at 20 h. For ascites, the i.p. route of administration resulted in high uptake (27.7 +/- 5.8%/g) early (2 h) with an ascites:normal tissue ratio of 69.3 and a specific antibody:nonspecific antibody ratio of 30.8. Except for data at one time point, uptake by i.p. solid tumor was similar to that seen for s.c. tumor. These data strongly suggest that large concentration advantages can be achieved in ascites cells by regional i.p. injection, but that i.p. solid tumor may rely on i.v. delivery of antibody before uptake.     

Analysis of production,purification, and cytolytic potential of bi-specific antibodies reactive with ovarian-carcinoma-associated antigens and the T-cell antigen CD3

OV-TL 3 and MOvl8 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might therefore be used to develop therapies for ovarian cancer. The bi-specific MAbs αT3/OC2 and αOC/TR (both being combinations of MOvl8 and aCD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOvl8/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs αT3/OC2 and αOC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were highly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma supernatants was analyzed by immunohistochemistry, and by testing bi-specific MAb-mediated cytotoxicity against relevant target cells in the presence of effector cells. Stability testing of bi-specific MAb-producing hybrid-hybridomas showed that, after sub-cloning, clones stably produced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with protein A and by ion-exchange high-pressure liquid chromatography, depending on the sub-class combination of the bi-specific MAb. The purified bi-specific MAbs were tested for their ability to mediate target-cell lysis with the use of cytotoxic T-cell clones and activated peripheral-blood lymphocytes. The purified αT3/OC2, αOC/TR, and OV-TL 3/CD3 were all able to mediate highly specific lysis of various ovarian-carcinoma cell lines. No correlation was found between the level of antigen expression and bi-specific MAb-mediated cytolysis. © 1993 Wiley-Liss, Inc.     

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.     

Radiolocalization of human pancreatic tumors in athymic mice by monoclonal antibody DU-PAN 1

Monoclonal antibodies that selectively bind to pancreatic tumors may be useful in the therapy and diagnosis of pancreatic carcinoma. In this study we have examined the tumor localization of radioiodinated DU-PAN 1, a mouse monoclonal antibody that is selective for a human pancreatic cancer-associated antigen. After radiolabeling, both DU-PAN 1 intact monoclonal antibody and F(ab')2 fragments retained immunoreactivity and showed high affinity for the pancreatic tumor cell line CA13 in vitro. Paired-label biodistribution studies in nude mice bearing CA13 s.c. xenografts were performed. Mice received both 131I-labeled DU-PAN 1 immunoglobulin G2a or F(ab')2 fragment and 125I-labeled mouse myeloma immunoglobulin G2a or F(ab')2 fragment. Tumor uptake for 5-micrograms doses of DU-PAN 1 immunoglobulin ranged from 4.8 to 11.83% injected dose/g. Tumor uptake values for mice given 5-micrograms doses of DU-PAN 1 F(ab')2 ranged from 3.9 to 6.9% injected dose/g. Tumor uptakes of the respective myeloma controls were lower in all cases when compared with the DU-PAN 1 preparations. Tumor localization indices for 5-micrograms doses of DU-PAN 1 immunoglobulin were 3.0 and 24 h and 2.9 at 48 h. For 5-micrograms doses of DU-PAN 1 F(ab')2, tumor localization indices were 29.9 at 24 h and 90.0 at 48 h. In most cases, tumor:normal tissue ratios were greater than 3 at all time points, indicative of tumor selectivity for both DU-PAN 1 preparations, but the ratios were considerably higher using the DU-PAN 1 F(ab')2. The F(ab')2 fragment thus displays better tumor localization characteristics when compared with the intact immunoglobulin. Protein doses of DU-PAN 1 F(ab')2 of between 5 and 10 micrograms gave the best localization, although protein doses of up to 100 micrograms could be administered before apparent tumor saturation was seen.     

Localization and imaging of radiolabelled monoclonal antibody against squamous-cell carcinoma of the head and neck in tumor-bearing nude mice

Nude mice carrying human squamous-cell carcinoma xenografts were given i.v. injections of radiolabelled monoclonal antibodies (MAbs). MAb E 48, which reacts with squamous-cell carcinomas, was labelled with 131I, while a second control MAb of similar immunoglobulin subclass was labelled with 125I. Both antibodies were injected simultaneously, then the mice were scanned with a gamma camera or their tissues were removed and antibody uptake was calculated as a percentage of the injected dose. Uptake of E 48 reached a peak value of 16%/g on day 3, while uptake of the control antibody was less than 1.8%/g. By 24 hr after injection tumor could be visualized without subtraction techniques. At days 3 and 7, only xenografts were visible on imaging. These findings suggest that E 48 is capable of high specificity in targeting isotopes to squamous-cell carcinomas in an experimental setting.     

Pharmacokinetics of 99mTc(Sn)- and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of 99mTc(Sn) and 131I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant 125I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake [48.6% injected dose (ID)/g +/- 8.1% (SD)] and low tumor uptake (1.5% ID/g +/- 0.6%) for 99mTc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for 131I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the 131I-labeled fragments and poor tumor visualization with predominant kidney uptake for 99mTc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.     

Biodistribution of monoclonal antibody A7 and its F(ab')2 fragment in athymic nude mice bearing human pancreatic carcinoma.

Xenografts of a human pancreatic carcinoma cell line, HPC-YS, which reacted with the monoclonal antibody (MAb) A7, were used to investigate the in vivo localization of radioiodinated MAb A7 after intraperitoneal injection. MAb A7 localized to the tumor 4 days and 8 days after injection with a tissue/blood ratio of 1.45 +/- 0.18 and 2.04 +/- 0.20, respectively. The accumulation of MAb A7 in the tumor was 5%/g and 3.3%/g of the injected dose on day 4 and on day 8, respectively. In contrast, the F(ab')2 fragment of MAb A7 localized to the tumor 4 days after intravenous injection with a tissue/blood ratio of 14.2. The accumulation of the F(ab')2 fragment in the tumor was 1.2%/g of the injected dose. These results suggested that MAb A7 might be a suitable carrier of anticancer drugs for immunotargeting chemotherapy and that the F(ab')2 fragment might be potentially useful for the immunodetection of human pancreatic carcinomas.     

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.     

Enhanced delivery of a monoclonal antibody F(ab')2 fragment to subcutaneous human glioma xenografts using local hyperthermia

The purpose of this study was to investigate the effects of tumor-localized hyperthermia at 42 degrees C on the tissue distribution of radioiodinated monoclonal antibody F(ab')2 fragments. Paired-label biodistribution measurements were performed in athymic mice bearing D-54 MG human glioma xenografts on one leg. Mice received both the 131I-labeled F(ab')2 fragment of Mel-14, reactive with human gliomas and melanomas, and nonspecific 125I-labeled RPC 5 F(ab')2. Tumor-bearing legs were placed in a 42 degrees C water bath or a 37 degrees C water bath (control) for 2 or 4 h. In mice sacrificed immediately after 2 h of heating, no hyperthermia-induced differences in the distribution of either fragment were observed. In the 4-h groups, tumor uptake of Mel-14 F(ab')2 increased from 7.04 +/- 1.59% injected dose (ID)/g at 37 degrees C to 20.65 +/- 4.53% ID/g at 42 degrees C (P less than 0.0001), and tumor localization of the control fragment rose from 5.23 +/- 1.35% ID/g to 14.51 +/- 1.37% ID/g (P less than 0.0001). In another experiment, F(ab')2 fragments were injected, tumors were heated for 4 h, and groups were sacrificed at 4, 8, and 16 h after injection. Statistically significant 2- to 3-fold higher uptake of both fragments in tumor were observed at all time points. Hyperthermic conditions also resulted in higher tumor:tissue ratios for both fragments. These results suggest that it may be possible to use tumor-localized hyperthermia to increase the therapeutic utility of radiolabeled monoclonal antibodies, particularly when labeled with short lived nuclides such as the 7.2-h alpha-emitter 211At.     

Radiohalogenation of a monoclonal antibody using an N-succinimidyl 3-(tri-n-butylstannyl)benzoate intermediate

N-Succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE) was elevated for its utility in the radiohalogenation of monoclonal antibodies. The F(ab')2 fragment of monoclonal antibody OC 125 was labeled with 125I using the ATE reagent and with 131I using a conventional electrophilic iodination method (Iodogen). N-Succinimidyl 3-[125I]iodobenzoate was synthesized from ATE in greater than 90% yield and purified using a disposable silica gel cartridge. About 60-65% of the radioiodinated product was coupled to the F(ab')2 fragment after a 30-min reaction. Two procedures were investigated, one involving exposure of antibody to 35 nmol of ATE and the other to 240 nmol of ATE. Using Scatchard analyses, affinity constants for binding to CA 125 antigen for OC 125 F(ab')2 labeled using the low ATE, Iodogen, and high ATE procedures were determined to be (5.2 +/- 1.0) x 10(10), (2.5 +/- 0.9) x 10(10), and (4.2 +/- 2.4) x 10(9) M-1, respectively. Paired-label studies in athymic mice bearing OVCAR-3 tumors treated with injections of antibody labeled via both ATE and Iodogen demonstrated that use of the ATE method (a) reduced thyroid uptake to less than 0.1% of the injected dose, more than 100 times less than that observed with Iodogen; (b) resulted in more rapid clearance of activity from normal tissues; and (c) with the low ATE preparations, increased the uptake of radioactivity in tumor from 27 to 49%. At 96 h, tumor:tissue ratios were generally at least 4-fold higher when antibody was labeled via ATE. These results suggest that the ATE method may be a valuable approach for the radiohalogenation of antibodies.