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Radioactive monoclonal antibodies against cell surface antigens for labeling leukocyte subpopulations
668
Radiolabeled MonoclonalAntibodies such as albumin can penetrate deeper than conventional chemotherapy agents of smaller molecular weight. Using an experimental model (nude mice with i.p. implanted human xenograft) it was shown that we could achieve a tenfold increase and faster localization of i.p. administered antibody as compared to i.v. administration. A Phase I clinical trial of i.p. administered I-131-labelled tumourassociated monoclonal antibodies was conducted in 12 patients with advanced ovarian cancer resistant to chemotherapy and/or abdominal radiotherapy. Toxicity was noted at a level of 100 mCi in patients with malignant ascites, and 150 mCi in patients without ascites. Diarrhoea and cytopenia were more marked in patients who had previously received abdominal radiotherapy. Three out of four patients with stage IV and large volume disease (>2 cm diameter) benefited symptomatically but died of their disease 3-6 months after treatment. Two patients with stage III and large volume disease (>2 cm diameter) had stable disease at 6 and 8 months after treatment. Four out of six patients with stage III and small volume (< 2 cm diameter) achieved complete remission for 3-24 months after treatment. Two out of these patients had positive washings as the only evidence of disease and they remain in remission for more than 2 years after therapy. A Phase II double blind randomised clinical trial has recently commenced to examine these effects on a larger number of patients and to define the mode of action of this method.
RADIOACTIVRmlNOCLONALANTIRODIRS AGAINST CRLLSDRFACEANTIGRNS PORLADRLING John G. McAfee, G. Gagne, and G. Subramanian. LRDROCYTR SWPOPDLATIONS. State University of New York Health Science Center, Syracuse, NY, USA. Hundreds of monoclonal antibodies (MAb) already have been developed against cell surface antigens of human leukocyte subpopulations. They have been used chiefly for cell identificationwith fluorescent-labeledsecondary antibody and flow cytometry, and for lysis of unwanted cells after the addition of rabbit complement. Some MAbs are specific for granulocytes, monocytes, B lymphocytes, T lymphocytes, helper T cells, or cytotoxic T cells. We have explored radioactive MAbs for labeling granulocytes and lymphocytes. The advantages of this approach include: 1) labeling is often satisfactory in plasma; 2) labeling is sometimes irreversible; 3) labeling is often cell-specific, thereby avoiding tedious physical methods of cell separation; and 4) radioactivity is confined to the cell membrane or cytoplasm, thereby minimizing self-irradiation damage to the cell nucleus from Auger electrons, particularly for radiation-sensitivelymphocytes. Unlike radioactive MAbs for in-vivo tumor localization, the antigens of the leukocyte cell surface are exposed directly to the MAbs in vitro. The difficulties encountered with this approach include: 1) the expense of commercial MAbs; 2) frequent contamination of "purified" MAb from murine ascitic fluid containing other proteins; 3) contamination of hybridoma supernatant with Ig from fetal calf serum; and 4) sodium aside preservative and aggregates of IgG must be eliminated. MAbs were labeled with I-131 by the Pierce Iodobead technique and with In-111 after coupling with the DTPA cyclic dianhydride, and unbound radioactivity removed by column chromatography. The labeled MAb (l-20 up) was added to 108 leukocytes (separated by gravity sedimentation) suspended in 0.5 ml plasma or saline and incubated for 1 h at room temperature. After cell washing, the cell labeling yield was measured. The cell suspensions were then washed in an elutriation centrifuge for 30 min to determine the stability of the cell label. Many MAbs proved unstable in their ability to label leukocytes, as revealed by elutriation. The best cell labeling yield was 25%, obtained with the use of 2 ug of MAb. Human granulocytes were irreversibly labeled with two IgM MAbs, BD anti-Leu M4 (clone G7-Eli) and NEI-044 (clone IGlO). Only one MAb has been found which irreversibly labels human T cells, NEI-016 (antibody 9.6, clone Lyt 31, an IgG2b. Thus, radioactive MAbs can be used for leukocyte labeling; however, a considerable number of MAbs will have to be screened to find the ones which bind firmly to leukocytes.
Radiolabeled MonoclonalAntibodies such as albumin can penetrate deeper than conventional chemotherapy agents of smaller molecular weight. Using an experimental model (nude mice with i.p. implanted human xenograft) it was shown that we could achieve a tenfold increase and faster localization of i.p. administered antibody as compared to i.v. administration. A Phase I clinical trial of i.p. administered I-131-labelled tumourassociated monoclonal antibodies was conducted in 12 patients with advanced ovarian cancer resistant to chemotherapy and/or abdominal radiotherapy. Toxicity was noted at a level of 100 mCi in patients with malignant ascites, and 150 mCi in patients without ascites. Diarrhoea and cytopenia were more marked in patients who had previously received abdominal radiotherapy. Three out of four patients with stage IV and large volume disease (>2 cm diameter) benefited symptomatically but died of their disease 3-6 months after treatment. Two patients with stage III and large volume disease (>2 cm diameter) had stable disease at 6 and 8 months after treatment. Four out of six patients with stage III and small volume (< 2 cm diameter) achieved complete remission for 3-24 months after treatment. Two out of these patients had positive washings as the only evidence of disease and they remain in remission for more than 2 years after therapy. A Phase II double blind randomised clinical trial has recently commenced to examine these effects on a larger number of patients and to define the mode of action of this method.
RADIOACTIVRmlNOCLONALANTIRODIRS AGAINST CRLLSDRFACEANTIGRNS PORLADRLING John G. McAfee, G. Gagne, and G. Subramanian. LRDROCYTR SWPOPDLATIONS. State University of New York Health Science Center, Syracuse, NY, USA. Hundreds of monoclonal antibodies (MAb) already have been developed against cell surface antigens of human leukocyte subpopulations. They have been used chiefly for cell identificationwith fluorescent-labeledsecondary antibody and flow cytometry, and for lysis of unwanted cells after the addition of rabbit complement. Some MAbs are specific for granulocytes, monocytes, B lymphocytes, T lymphocytes, helper T cells, or cytotoxic T cells. We have explored radioactive MAbs for labeling granulocytes and lymphocytes. The advantages of this approach include: 1) labeling is often satisfactory in plasma; 2) labeling is sometimes irreversible; 3) labeling is often cell-specific, thereby avoiding tedious physical methods of cell separation; and 4) radioactivity is confined to the cell membrane or cytoplasm, thereby minimizing self-irradiation damage to the cell nucleus from Auger electrons, particularly for radiation-sensitivelymphocytes. Unlike radioactive MAbs for in-vivo tumor localization, the antigens of the leukocyte cell surface are exposed directly to the MAbs in vitro. The difficulties encountered with this approach include: 1) the expense of commercial MAbs; 2) frequent contamination of "purified" MAb from murine ascitic fluid containing other proteins; 3) contamination of hybridoma supernatant with Ig from fetal calf serum; and 4) sodium aside preservative and aggregates of IgG must be eliminated. MAbs were labeled with I-131 by the Pierce Iodobead technique and with In-111 after coupling with the DTPA cyclic dianhydride, and unbound radioactivity removed by column chromatography. The labeled MAb (l-20 up) was added to 108 leukocytes (separated by gravity sedimentation) suspended in 0.5 ml plasma or saline and incubated for 1 h at room temperature. After cell washing, the cell labeling yield was measured. The cell suspensions were then washed in an elutriation centrifuge for 30 min to determine the stability of the cell label. Many MAbs proved unstable in their ability to label leukocytes, as revealed by elutriation. The best cell labeling yield was 25%, obtained with the use of 2 ug of MAb. Human granulocytes were irreversibly labeled with two IgM MAbs, BD anti-Leu M4 (clone G7-Eli) and NEI-044 (clone IGlO). Only one MAb has been found which irreversibly labels human T cells, NEI-016 (antibody 9.6, clone Lyt 31, an IgG2b. Thus, radioactive MAbs can be used for leukocyte labeling; however, a considerable number of MAbs will have to be screened to find the ones which bind firmly to leukocytes.