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Considerations in the diagnosis and therapy of human T-cell lymphoma with monoclonal antibodies
184
Abstracts
associated antigen. The antibodies were purified from the ascitic fluid of mice, bearing hybridoma ascites tumor, using affinity chromatography on protein A-Sepharose. The Fab fragments were then prepared by papain digestion. The Fabs were radioiodinated using the chloramine-T method. All products were then tested for immunoreactivity, sterility and pyrogens. The pharmacokinetics of the Fab fragments was different from that of IgG. The Fab had a faster plasma clearance and a more rapid whole body excretion than the IgG. Using the biodistribution data from the diagnostic studies and concepts outlined by the MIRD committee, dosimetry estimates were calculated. It was estimated that for 100 mCi of 13’1-Fab the whole body dose was 39 rem and the marrow dose was 30 rem. Twenty one patients with advanced metastatic melanoma received a therapeutic dose of 13’1-Fab that ranged from a single dose of 34-342 mCi as part of a phase 1 therapy trial. Four of the initial IO patients treated received more than one therapeutic dose separated by at least a 2-3 week period. Due to the advanced status of the patients’ disease 6 patients could not receive a full course of therapy. One patient had partial regression of involved inguino-femoral lymph nodes after two therapy doses totaling 347 mCi of 13’1-Fab 48.7. Further treatment could not be given because of human anti-mouse antibodies. One patient appeared to have a decrease in the growth rate of his tumor. Eleven patients were recruited to receive the three therapy doses over a shorter (2 week) period. Due to the advanced nature of the patients metastatic disease 6 patients received three therapy doses, 3 patients received two therapy doses (within 7 days) and 2 patients received only one therapy dose. In this group no therapeutic responses were visualized. While one patient had a decrease in the growth rate of his tumors and another had a decrease in pain medication, no objective tumor responses were visualized. While some patients developed an anti-murine response and a rash after multiple injections the main toxicity was hematological. Weekly administration of 13’1-Fab were more toxic than those performed with longer intervals between doses. All three patients receiving >450 mCi within a 2 week period developed moderate to life threatening hematological toxicity. The nadir was usually within 6 weeks. Where the follow up is available, patients recuperated marrow function spontaneously. One patient developed a slow GI bleed from pre-existing peptic ulcer disease and another patient develop a gastric perforation. The latter two complications may have been coincidental and not related to the radiolabeled antibody. In conclusion we have; (1) labeled Fab fragments with large doses of 13’1and have been able to document tumor uptake; (2) in the absence of human anti-mouse immune response, the 13’I-Fab can be administered repetitively and produce good tumor reproducible tumor uptake; (3) weekly administration of high doses of 13’I-Fab are more toxic than separating the doses by 2-3 weeks; (4) with exception of one partial regression no objective tumor responses were visualized. In order to improve the therapeutic index methods to enhance the tumor uptake will be needed.
Considerations in the Diagnosis and Therapy of Human T-cell Lymphoma with Monoclonal Antibodies JORGE A. CARRASQUILLO, PAUL A. BUNN, JAMES C. MULSHINE, KENNETH A. FOON, JAMES C. REYNOLDS, ROBERT W. SCHROFF, PATRICIA PERENTESIS and STEVEN M. LARSON Department of Nuclear Medicine, National Institutes of Health, Bethesda, Md, U.S.A.
Patients with lymphomas must undergo a thorough diagnostic staging work up to define the extent of their disease, their prognosis and to determine the optimum therapy. The currently available techniques involve a series of non-invasive radiographic procedures including plain films, CT scans, MRI, gallium scans and lymphangiogram. All these tests provide indirect evidence of the presence of tumor since they depend on non-specific differences in attenuation, ultrasound reflection, paramagnetic properties or metabolism. These tests are often not sufficient for adequate staging and the patients must undergo a staging laparotomy. The field of radioimmunoscintigraphy (RIS) offers the potential of greater specificity by using radiolabeled anti-tumor antibodies as probes for tumor detection. Most of the antigens identified for targeting lymphomas are not tumor specific, but there is a quantitative difference in expression of these antigens between normal and tumor tissue. We have successfully targeted tumor sites in patients with cutaneous T-cell lymphoma (CTCL) using TlOl, a murine monoclonal antibody (MoAb). TlOl is a IgG2a that recognizes CD5, a pan T-cell cell surface antigen that undergoes antigenic modulation. The CD5 is present on normal T-lymphocytes, T-cell lymphomas including CTCL and also expressed on chronic lymphocytic leukemia. The antigen concentration on CTCL cells is much higher than on normal T-lymphocytes. TlOl was purified from hybridoma ascites of Balb/c mice by precipitation with 18% Na sulfate and DEAE-Sephacel column chromatography. The antibody was conjugated to diethylentriaminepentaacetic acid (DTPA) using a modification of the mixed anhydride method. The conjugated TIOI was then labeled with “‘In at specific activities of approximately 88% as tested by a cell binding assay.
Abstracts
I85
Groups of patients received iv. injection of a fixed dose of “‘In-T101 (5mCi and 1 mg) alone or mixed with 10 mg or 50 mg of unlabeled TlOl. To determine the pharmacokinetics, serial blood clearance and whole body retention measurements were performed. Scintillation camera images were recorded with a large field-of-view y camera within 2 h post infusion, and after 24, 48, 72 h and at later times up to 1 week. The images were acquired with a nuclear medicine y camera using a medium energy collimator utilizing separate 20% windows centered over the 173 and 247 keV photopeaks. The scan data were stored on a nuclear medicine computer and analyzed by manually drawing regions of interest over the organs of interest. In the initial 11 patients studied there as marked concentration of “‘In-T101 in lymph nodes that had been shown pathologically or clinically to be involved, as well as in many clinically unsuspected nodal sites. The scan was positive in 38 of 39 clinically involved nodal regions. An additional 44 clinically negative sites showed “‘In-T101 concentration. Four of these sites were confirmed true positive by biopsy, while the other 40 were not biopsied and therefore not confirmed. Areas of cutaneous involvement with erythroderma or tumors were positive while skin plaques were not visualized, perhaps related to the lower number of malignant T-cells in skin plaques. Control studies with “‘In alone or a “‘In control (anti-melanoma) antibody did not show tumor localization. The total antibody mass did not affect the concentration of radioactivity in tumor sites or the number of lesions visualized. However there were significant differences in the biodistribution in normal tissue (liver, spleen and bone marrow). When 1 mg was infused there was rapid removal from the circulation with only 10 + 3% of the injected radioactivity in the circulation 2 h after the end the infusion. When 10 or 50 mg was infused 58 + 10% of the injected activity was in the blood pool 2 h after infusion. At all doses localization was seen in lymph nodes by 24 h. In addition to the concentration in involved nodes, all patients had a high concentration in liver, spleen and bone marrow. The mechanism of uptake in these organs is likely to be multifactorial. The patients studied showed a rapid decline in the circulating lymphocytes. TlOl binds to circulating CD5 positive cells which are then cleared predominantly into the liver. A small amount of “‘In-transferrin was found in the circulation but was not enough to account for the liver uptake. The catabolism of immunoglobulin in the liver with retention of the “‘In may have contributed to the liver uptake. Skin biopsies performed after “‘In-T101 infusion showed a concentration that ranged from 3.4 to 44 x 10d4% of the injected dose per gram with a trend toward higher concentration in the patients with more extensive skin involvement. Lymph node biopsy in three patients showed a range of 0.011-0.033% of the injected dose per gram, that is significantly higher than the values reported for other MoAb. “‘In us ‘3’I-TIOl
Using the methods outlined previously we evaluated the biodistribution of ‘3’I-T101 and compared this to that of “‘In-TlOl. The TlOl was labeled at specific activities of approximately 2mCi/mg using the chloramine-T method. The immunoreactivity for both products were similar: ‘3’I-T101 ranged from 74 to 77% while that of “IIn was 7690%. The ‘3’I-T101 resulted in suboptimal images with minimal or no localization in tumor sites. The initial ‘3’I-T101 images showed prominent accumulation in the liver and spleen and faint bone marrow uptake. The region of interest analysis showed rapid clearance of the radioactivity from the whole body, liver and spleen. The predominant route of excretion was urinary. The biologic half-life of clearance from the whole body was 27 h for 13’1and > 7 days for “‘In-TlOl. A comparison of the biodistribution of j3’I and “‘In-T101 in the same patient confirmed these differences. In contrast the plasma clearance of the 13’1and “‘In-T101 was not significantly different, suggesting that the differences occured after the antibody left the circulation. Previous in vitro and in vivo work has shown that CD5 antigen modulates quickly in the presence of TlOl and becomes internalized. Our studies suggest that while both antibody preparations behaved similarly initially, once internalization occurred the antibody was likely to be broken down and 13’1was rapidly released whereas “‘In was trapped intracellularly. In vitro studies have shown that ‘2sI-T101 will bind to antigen bearing cells and lzsI will be rapidly released from the cells in the form of non protein bound ‘251.The “‘In will also bind to antigen bearing cells but in contrast to the iodinated TlOl it will be retained for a longer suggest that once 9 binds to antigen bearing period of time. An in vitro comparison of “‘In and v-T101 cells it will behave more like ‘*‘In with prolonged retention rather than the rapid breakdown and release seen with iodine-TlOl. These studies indicate that, although the antibodies may be labeled successfully with different radiolabels the in vivo handling of the antibody and/or the isotope may be drastically different and result in large differences in biodstribution. This studies point out the advantage of “‘In over iodine and suggest that other radionuclides may be more favorable than iodine for RIT with TlOl, in contrast other isotopes, that are retained by the cells after targeting, such as 9oy may be more useful for the treatment of CTCL. As a result a phase 1 therapy trial will be conducted utilizing ?‘-TlOl to evaluate this hypothesis.
Abstracts
associated antigen. The antibodies were purified from the ascitic fluid of mice, bearing hybridoma ascites tumor, using affinity chromatography on protein A-Sepharose. The Fab fragments were then prepared by papain digestion. The Fabs were radioiodinated using the chloramine-T method. All products were then tested for immunoreactivity, sterility and pyrogens. The pharmacokinetics of the Fab fragments was different from that of IgG. The Fab had a faster plasma clearance and a more rapid whole body excretion than the IgG. Using the biodistribution data from the diagnostic studies and concepts outlined by the MIRD committee, dosimetry estimates were calculated. It was estimated that for 100 mCi of 13’1-Fab the whole body dose was 39 rem and the marrow dose was 30 rem. Twenty one patients with advanced metastatic melanoma received a therapeutic dose of 13’1-Fab that ranged from a single dose of 34-342 mCi as part of a phase 1 therapy trial. Four of the initial IO patients treated received more than one therapeutic dose separated by at least a 2-3 week period. Due to the advanced status of the patients’ disease 6 patients could not receive a full course of therapy. One patient had partial regression of involved inguino-femoral lymph nodes after two therapy doses totaling 347 mCi of 13’1-Fab 48.7. Further treatment could not be given because of human anti-mouse antibodies. One patient appeared to have a decrease in the growth rate of his tumor. Eleven patients were recruited to receive the three therapy doses over a shorter (2 week) period. Due to the advanced nature of the patients metastatic disease 6 patients received three therapy doses, 3 patients received two therapy doses (within 7 days) and 2 patients received only one therapy dose. In this group no therapeutic responses were visualized. While one patient had a decrease in the growth rate of his tumors and another had a decrease in pain medication, no objective tumor responses were visualized. While some patients developed an anti-murine response and a rash after multiple injections the main toxicity was hematological. Weekly administration of 13’1-Fab were more toxic than those performed with longer intervals between doses. All three patients receiving >450 mCi within a 2 week period developed moderate to life threatening hematological toxicity. The nadir was usually within 6 weeks. Where the follow up is available, patients recuperated marrow function spontaneously. One patient developed a slow GI bleed from pre-existing peptic ulcer disease and another patient develop a gastric perforation. The latter two complications may have been coincidental and not related to the radiolabeled antibody. In conclusion we have; (1) labeled Fab fragments with large doses of 13’1and have been able to document tumor uptake; (2) in the absence of human anti-mouse immune response, the 13’I-Fab can be administered repetitively and produce good tumor reproducible tumor uptake; (3) weekly administration of high doses of 13’I-Fab are more toxic than separating the doses by 2-3 weeks; (4) with exception of one partial regression no objective tumor responses were visualized. In order to improve the therapeutic index methods to enhance the tumor uptake will be needed.
Considerations in the Diagnosis and Therapy of Human T-cell Lymphoma with Monoclonal Antibodies JORGE A. CARRASQUILLO, PAUL A. BUNN, JAMES C. MULSHINE, KENNETH A. FOON, JAMES C. REYNOLDS, ROBERT W. SCHROFF, PATRICIA PERENTESIS and STEVEN M. LARSON Department of Nuclear Medicine, National Institutes of Health, Bethesda, Md, U.S.A.
Patients with lymphomas must undergo a thorough diagnostic staging work up to define the extent of their disease, their prognosis and to determine the optimum therapy. The currently available techniques involve a series of non-invasive radiographic procedures including plain films, CT scans, MRI, gallium scans and lymphangiogram. All these tests provide indirect evidence of the presence of tumor since they depend on non-specific differences in attenuation, ultrasound reflection, paramagnetic properties or metabolism. These tests are often not sufficient for adequate staging and the patients must undergo a staging laparotomy. The field of radioimmunoscintigraphy (RIS) offers the potential of greater specificity by using radiolabeled anti-tumor antibodies as probes for tumor detection. Most of the antigens identified for targeting lymphomas are not tumor specific, but there is a quantitative difference in expression of these antigens between normal and tumor tissue. We have successfully targeted tumor sites in patients with cutaneous T-cell lymphoma (CTCL) using TlOl, a murine monoclonal antibody (MoAb). TlOl is a IgG2a that recognizes CD5, a pan T-cell cell surface antigen that undergoes antigenic modulation. The CD5 is present on normal T-lymphocytes, T-cell lymphomas including CTCL and also expressed on chronic lymphocytic leukemia. The antigen concentration on CTCL cells is much higher than on normal T-lymphocytes. TlOl was purified from hybridoma ascites of Balb/c mice by precipitation with 18% Na sulfate and DEAE-Sephacel column chromatography. The antibody was conjugated to diethylentriaminepentaacetic acid (DTPA) using a modification of the mixed anhydride method. The conjugated TIOI was then labeled with “‘In at specific activities of approximately 88% as tested by a cell binding assay.
Abstracts
I85
Groups of patients received iv. injection of a fixed dose of “‘In-T101 (5mCi and 1 mg) alone or mixed with 10 mg or 50 mg of unlabeled TlOl. To determine the pharmacokinetics, serial blood clearance and whole body retention measurements were performed. Scintillation camera images were recorded with a large field-of-view y camera within 2 h post infusion, and after 24, 48, 72 h and at later times up to 1 week. The images were acquired with a nuclear medicine y camera using a medium energy collimator utilizing separate 20% windows centered over the 173 and 247 keV photopeaks. The scan data were stored on a nuclear medicine computer and analyzed by manually drawing regions of interest over the organs of interest. In the initial 11 patients studied there as marked concentration of “‘In-T101 in lymph nodes that had been shown pathologically or clinically to be involved, as well as in many clinically unsuspected nodal sites. The scan was positive in 38 of 39 clinically involved nodal regions. An additional 44 clinically negative sites showed “‘In-T101 concentration. Four of these sites were confirmed true positive by biopsy, while the other 40 were not biopsied and therefore not confirmed. Areas of cutaneous involvement with erythroderma or tumors were positive while skin plaques were not visualized, perhaps related to the lower number of malignant T-cells in skin plaques. Control studies with “‘In alone or a “‘In control (anti-melanoma) antibody did not show tumor localization. The total antibody mass did not affect the concentration of radioactivity in tumor sites or the number of lesions visualized. However there were significant differences in the biodistribution in normal tissue (liver, spleen and bone marrow). When 1 mg was infused there was rapid removal from the circulation with only 10 + 3% of the injected radioactivity in the circulation 2 h after the end the infusion. When 10 or 50 mg was infused 58 + 10% of the injected activity was in the blood pool 2 h after infusion. At all doses localization was seen in lymph nodes by 24 h. In addition to the concentration in involved nodes, all patients had a high concentration in liver, spleen and bone marrow. The mechanism of uptake in these organs is likely to be multifactorial. The patients studied showed a rapid decline in the circulating lymphocytes. TlOl binds to circulating CD5 positive cells which are then cleared predominantly into the liver. A small amount of “‘In-transferrin was found in the circulation but was not enough to account for the liver uptake. The catabolism of immunoglobulin in the liver with retention of the “‘In may have contributed to the liver uptake. Skin biopsies performed after “‘In-T101 infusion showed a concentration that ranged from 3.4 to 44 x 10d4% of the injected dose per gram with a trend toward higher concentration in the patients with more extensive skin involvement. Lymph node biopsy in three patients showed a range of 0.011-0.033% of the injected dose per gram, that is significantly higher than the values reported for other MoAb. “‘In us ‘3’I-TIOl
Using the methods outlined previously we evaluated the biodistribution of ‘3’I-T101 and compared this to that of “‘In-TlOl. The TlOl was labeled at specific activities of approximately 2mCi/mg using the chloramine-T method. The immunoreactivity for both products were similar: ‘3’I-T101 ranged from 74 to 77% while that of “IIn was 7690%. The ‘3’I-T101 resulted in suboptimal images with minimal or no localization in tumor sites. The initial ‘3’I-T101 images showed prominent accumulation in the liver and spleen and faint bone marrow uptake. The region of interest analysis showed rapid clearance of the radioactivity from the whole body, liver and spleen. The predominant route of excretion was urinary. The biologic half-life of clearance from the whole body was 27 h for 13’1and > 7 days for “‘In-TlOl. A comparison of the biodistribution of j3’I and “‘In-T101 in the same patient confirmed these differences. In contrast the plasma clearance of the 13’1and “‘In-T101 was not significantly different, suggesting that the differences occured after the antibody left the circulation. Previous in vitro and in vivo work has shown that CD5 antigen modulates quickly in the presence of TlOl and becomes internalized. Our studies suggest that while both antibody preparations behaved similarly initially, once internalization occurred the antibody was likely to be broken down and 13’1was rapidly released whereas “‘In was trapped intracellularly. In vitro studies have shown that ‘2sI-T101 will bind to antigen bearing cells and lzsI will be rapidly released from the cells in the form of non protein bound ‘251.The “‘In will also bind to antigen bearing cells but in contrast to the iodinated TlOl it will be retained for a longer suggest that once 9 binds to antigen bearing period of time. An in vitro comparison of “‘In and v-T101 cells it will behave more like ‘*‘In with prolonged retention rather than the rapid breakdown and release seen with iodine-TlOl. These studies indicate that, although the antibodies may be labeled successfully with different radiolabels the in vivo handling of the antibody and/or the isotope may be drastically different and result in large differences in biodstribution. This studies point out the advantage of “‘In over iodine and suggest that other radionuclides may be more favorable than iodine for RIT with TlOl, in contrast other isotopes, that are retained by the cells after targeting, such as 9oy may be more useful for the treatment of CTCL. As a result a phase 1 therapy trial will be conducted utilizing ?‘-TlOl to evaluate this hypothesis.