Human sarcoma-associated murine monoclonal antibody labeled with indium-111, gallium-67, and iodine-125

Monoclonal antibody (MAb) 23H7 is a hybridoma-derived IgG that is generated following fusion of mouse myeloma cell line P3U1 and spleen cells from BALB/c mice hyperimmunized with a human fibrosarcoma. It detects a mesenchymal antigen of 23KD expressed on human sarcoma tissues and other neoplasms, including myeloid leukemias, but it rarely binds to normal tissues. The MAb 23H7 was labeled with 67Ga and 111In using the bifunctional ligand method. The 67Ga was chelated to the MAb via desferrioxamine B, while 111In was chelated via the cyclic anhydride of diethylenetriamine pentaacetic acid. Higher specific activity was obtained with 67Ga than with 111In (4.5 and 2 muCi/microgram, respectively); both gave stable complexes. When 23H7 was labeled with 125I, considerable breakdown was observed. This, together with the physical shortcomings of this isotope, emphasizes the advantages of labeling with 111In and 67Ga. The rapid blood clearance of the labeled sarcoma-associated MAb may be beneficial for early tumor uptake and for imaging shortly after injection.     

Gallium-67 radiotoxicity in human U937 lymphoma cells.

Promising clinical results have been obtained with radiolabeled antibodies in lymphoma patients. The higher uptake by lymphomas of 67Gallium (67Ga) compared with monoclonal antibodies makes selective radiotherapy by the widely available 67Ga appealing. However, the gamma radiation of 67Ga used in scintigraphy is considered to be almost non-toxic to lymphoma cells. However, in addition to photon radiation 67Ga emits low energy Auger electrons and 80-90 keV conversion electrons which could be cytotoxic. The objective of the present study was the assessment of radiotoxicity of 67Ga on a lymphoid cell line: U937. Proliferation (MTT-assay) and clonogenic capacity (CFU-assay) were measured after 3 and 6 days incubation with 10, 20 and 40 microCi ml-1 67Ga. Growth inhibition was 36% after 3 days incubation and 63% after 6 days incubation with 40 microCi 67Ga ml-1. Clonogenic capacity was reduced by 51% after 3 days and 72% after 6 days incubation with 40 microCi ml-1 67Ga. A survival curve showed an initial shoulder and became steeper beyond 200-250 pCi cell-1 (low linear energy transfer type). Iso-effect doses of 67Ga and 90Yttrium (90Y) were determined. The iso-effect dose of 40 microCi 67Ga ml-1 (cumulative dose of conversion electrons 306 cGy) was 2.5 microCi 90Y ml-1 (cumulative dose 494 cGy) and the iso-effect dose of 80 microCi 67Ga ml-1 was 5.0 microCi 90Y/ml. The main cytotoxic effect of 67Ga seems to be induced by the 80 keV conversion electrons. We conclude that the conversion electrons of 67Ga have a cytotoxic effect on U937 cells and that in our experiments a 16-fold higher microCi-dose of 67Ga than of 90Y was needed for the same cytotoxic effect. We believe that 67Ga holds promise for therapeutic use.     

High-dose gallium-67 therapy in patients with relapsed acute leukaemia: a feasibility study.

Gallium-67 (67Ga) accumulates in malignant tissues via the transferrin receptor without need for a monoclonal antibody and emits cytotoxic low-energy electrons. In this study we investigated the feasibility, pharmacokinetics, toxicity and preliminary efficiency of high-dose 67Ga injected intravenously (i.v.) in patients with acute leukaemia not responding to conventional therapy. Twelve doses of 36-105 mCi of Gallium67 citrate were administered as a push injection to eight patients with resistant leukaemia in a pilot study. All five patients with acute myeloid leukaemia (AML) and three patients with acute lymphoblastic leukaemia (ALL) had resistant disease or resistant relapse. No (sub)acute toxicity was observed. Independent of the administered dose, whole-blood radioactivity levels 10 min after administration measured only 1.25 +/- 1.39 microCi ml-1, indicating a large volume of distribution. Urine excretion in the first 24 h ranged from 18% to 51.5% (median 29.5%) of the administered dose. Cellular uptake of 67Ga was less than in previous in vitro studies. Whole-body radiation dose was estimated to be 0.25 +/- 0.03 cGy mCi-1. Red marrow dose was estimated to be between 0.18 +/- 0.02 and 0.97 +/- 0.12 cGy mCi-1. One definite response was observed in an ALL patient with disappearance of skin lesions, normalisation of the enlarged spleen and profound leucopenia. Three other patients showed transient reductions in white blood cell counts without disappearance of blasts from the peripheral blood. We conclude that high-dose i.v. 67Ga can be safely administered but that the uptake of 67Ga in blast cells must increase to make 67Ga therapeutically useful in patients with relapsed leukaemia.     

Evaluation of L6, an anti-carcinoma murine monoclonal antibody, in tumor-bearing nude mice

L6 is a murine monoclonal antibody (MAb) binding to cells of most human carcinomas, mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and inhibiting tumor growth in nude mice [10]. Fab and F(ab')2 fragments of L6, as well as intact MAb, have been evaluated for immunospecific localization in nude mice xenografted with a human lung carcinoma. They were compared with preparations of an isotype-matched control immunoglobulin, P1.17, after labelling with 125I or 131I. L6 Fab fragments prepared from MAb L6 and labelled with 67Ga via desferrioxamine were also tested. The data suggest that MAb L6 may be useful for in vivo detection of human carcinomas.     

A novel derivative of the chelon desferrioxamine for site-specific conjugation to antibodies

We describe the preparation of the modified chelator aminooxyacetyl-ferrioxamine, and the replacement of its iron atom by 67Ga at high specific activity. The aminooxy function of this compound was allowed to react with the aldehyde groups generated by the periodate oxidation of the oligosaccharide of a mouse IgG1 monoclonal antibody (MAb) directed against carcino-embryonic antigen (CEA). The use of the aminooxy group allowed a stable bond to be formed between the chelon and the antibody with no need for reduction. Iron was removed from the ferrioxamine moiety and replaced by 67Ga either before or after conjugation of the chelon to the antibody. In either case the labelled antibody was injected into nude mice bearing a human colon carcinoma having the appropriate antigenicity. Unoxidized antibody, labelled with 125I by conventional methods, was co-injected as an internal control. Additional control experiments were carried out with a non-immune IgG using the same 67Ga-labelled modified chelon as above. The in vivo distribution of the modified antibodies was evaluated at various times between 24 and 96 hr after injection. The methods used were gamma-camera imaging and, more quantitatively, gamma-counting of the various organs after dissection. Interestingly, with the metal-chelon-labelled antibody, the intensity and specificity of tumor labelling was comparable and in some cases superior to the results obtained with radio-iodinated antibody. In particular, there was almost no increase in liver and spleen uptake of radioactive metal relative to radio-iodine, contrary to what has been observed with most antibodies labelled with 111In after conjugation with DTPA.     

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.     

Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.

Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500-2000 microCi) or 131I-labeled CC49 IgG (250 and 500 microCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 microCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 microCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 microCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.     

Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells. In this study the F(ab')2 was the best compromise between the slowly cleared IgG and the poorly localized Fab in tumor imaging.     

Biodistribution and histological localization of anti-human colon cancer monoclonal antibody (MAb) 1A3: the influence of administered MAb dose on tumor uptake

Using a murine monoclonal antibody MAb 1A3, which binds to a lipid antigen found enriched in human colon cancer, and MOPC-21 antibody, as a non-specific control, we examined the effect of increasing doses of 125I-labelled MAbs (5 micrograms to 2,000 micrograms) on tumor localization. Biodistribution studies in hamsters with small GW-39 human colon carcinoma tumors [0.44 g +/- 0.014 (SEM)] demonstrated functional saturation of antigen binding sites in tumors when large doses of MAb 1A3 were used. The percentage injected dose bound per gram of tumor (ID/g tumor) remained constant for doses of intact MAb 1A3 less than or equal to 100 micrograms but decreased with doses greater than 100 micrograms, suggesting that a population of antigen binding sites had been saturated. While the percentage ID/g tumor decreased for doses of MAb 1A3 greater than 100 micrograms, the absolute amount specifically bound/g turnover increased (up to the 1,000 micrograms dose), suggesting that another population of less accessible 1A3 antigen could continue to bind MAb 1A3. In contrast, MOPC demonstrated a relatively constant percentage ID/g of tumor (2.26% +/- 0.11) throughout the dose range which was 2-3 times lower than MAb 1A3 (6.8% +/- 0.14) at plateau doses (5-100 micrograms). These data suggest that specific saturation of tumor binding sites was a biphasic phenomenon. Blood and normal tissues did not show binding kinetics suggesting saturation. Results of dose response experiments using MAb 1A3 F(ab')2 fragments (5 micrograms to 1,000 micrograms) closely paralleled those obtained with intact MAb 1A3, but with lower percentages of ID/g tumor, blood and non-tumor tissues as in previous studies with MAb 1A3 and other MAb F(ab')2 fragments. Histological examinations demonstrated that non-specific binding of MOPC or MAb 1A3 to tumor or normal tissues was of such low affinity as to be largely undetected after histological procedures. In contrast, MAb 1A3 (but not MOPC) showed specific cell-binding patterns to tumor but not normal tissues at all doses.     

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.     

The mechanism of hepatic uptake of a radiolabelled monoclonal antibody.

Clinical and experimental scintigraphic studies have found that radiolabelled antibodies are not only taken up by tumour(s) but also by normal liver. The accumulation of radionuclides in this organ poses a major problem to the use of radiolabelled antibodies as diagnostic and therapeutic tools. In an attempt to understand the mechanism of hepatic uptake and clearance of radiolabelled antibodies, the intrahepatic biodistribution of an 111In-labelled MAb (HMFG1), was determined following i.v. administration to normal male rats. Two hours after administration the liver contained 15% of the injected dose, with most of the remaining radioactivity in the blood. The hepatic burden of the 111In MAb remained constant over the next 72 hr in the face of decreasing blood levels of radioactivity as well as its urinary and faecal excretion. At 2 and 72 hr after injection, 50% and 10% respectively of the hepatic radiolabel was due to blood borne antibody. Following a collagenase-cell isolation procedure, only 23% of the amount remaining in the liver at 2 hr was found to be cell-associated; 66% was lost during the cell isolation and purification procedure. Cellular uptake increased with time so that, by 72 hr after administration, 58% was cell-associated and 29% freely removable. At all timepoints, the parenchymal cells contained more activity than non-parenchymal cells. No evidence of antibody-receptor interactions could be obtained either in vivo or in cultures of hepatic parenchymal and non-parenchymal cells. Our data suggest that the bulk of the hepatic burden of 111In MAb results from extravascular pooling of the antibody.     

Radioimmunotherapy of intracerebral human glioma xenografts with 131I-labeled F(ab')2 fragments of monoclonal antibody Mel-14

The administration of radiolabeled monoclonal antibodies to improve the treatment of malignant gliomas is dependent upon achieving effective tumor radiation dose while sparing normal tissues. We have evaluated the efficacy of 131I-labeled F(ab')2 fragment of monoclonal antibody Mel-14, an IgG2a reactive with the chondroitin sulfate proteoglycan antigen of gliomas, melanomas, and other neoplasms, in prolonging survival of athymic mice transplanted intracerebrally with D-54 MG human glioma xenografts. Studies indicated that in vitro immunoreactivity, affinity, and tumor localization in vivo of radiolabeled Mel-14 F(ab')2 were maintained at specific activities of 10-13 microCi/micrograms. Intravenous injection of 1500 microCi/115 micrograms or 2000 microCi/154 micrograms 131I-labeled Mel-14 F(ab')2 into mice 6-7 days after xenograft implantation resulted in significant survival prolongation over control animals (P = 0.009 using Wilcoxon rank sum analysis). In another experiment, 1500 microCi/126 micrograms 131I-labeled Mel-14 F(ab')2 improved survival significantly over controls (P = 0.006), while 1500 microCi/220 micrograms 131I-labeled nonspecific antibody did not (P = 0.2). Increasing the injected radiation dose to 3000 microCi 131I-labeled Mel-14 F(ab')2 did not significantly increase survival in tumor-bearing mice, because of supervening radiation toxicity. However, giving 3000 microCi 131I-labeled Mel-14 F(ab')2 in two doses of 1500 microCi, 48 h apart, did significantly prolong animal survival over controls (P = 0.001). Estimated radiation dose to tumor was 915 rad after injection of 3000 microCi 131I-labeled Mel-14 F(ab')2 in two doses, a dose higher than that delivered to normal tissues. The results of this study suggest that radiolabeled Mel-14 F(ab')2 be evaluated as an agent for radioimmunotherapy trials.     

Monoclonal antibody-based therapy of a human tumor xenograft with a 177lutetium-labeled immunoconjugate.

177Lutetium (177Lu) is a member of the family of elements known as lanthanides or rare earths. Monoclonal antibody (MAb) CC49, a murine IgG1, which is reactive with the tumor-associated antigen, TAG-72, has been shown previously to react with a wide range of human carcinomas; CC49 reacts to a different epitope on the TAG-72 molecule than MAb B72.3 and has a higher binding affinity. We report here the first use of a 177Lu-labeled immunoconjugate, 177Lu-CC49, in an experimental therapy model for human carcinoma. 177Lu-CC49 was shown to delay the growth of established LS-174T human colon carcinomas in athymic mice at a single dose of 50 microCi. Overt toxicity was observed with the administration of approximately 500 microCi of 177Lu-CC49 in which 5 of 9 mice died of apparent marrow toxicity. A single administration of 200 or 350 microCi of 177Lu-CC49, however, was shown to eliminate established tumors through the 77-day observation period after MAb administration. Dose fractionation experiments revealed that at least 750 microCi of 177Lu-CC49 (250 microCi/week for 3 consecutive weeks) was well tolerated in that 9 of 10 mice survived. Moreover, this dose schedule was able to eliminate the growth of relatively large (300 mm3) human colon tumor xenografts in 90% of the animals treated. Single-dose and dose fractionation studies were also carried out with an isotype-matched control MAb, 177Lu-MOPC-21. In all dose schedules, a large differential was seen between the therapeutic effects of the 177Lu-CC49 versus that of the 177Lu-control MAb. The merits and limitations of the use of 177Lu-labeled immunoconjugates (in particular, 177Lu-CC49) are discussed in terms of potential novel therapeutics for human carcinoma.     

Therapy of disseminated B-cell lymphoma xenografts in severe combined immunodeficient mice with an anti-CD74 antibody conjugated with (111)indium, (67)gallium, or (90)yttrium.

A radiolabeled antibody (Ab) to CD74 (the MHC class II invariant chain, Ii) was shown previously to effectively kill human B-lymphoma cells in vitro. Conjugates with both Auger electron and beta-particle emitters were able to kill cells, but the former displayed less nonspecific toxicity in the in vitro assay used. In this report, we have extended the studies to an in vivo model of tumor growth. The human B-cell lymphoma Raji was injected i.v. into severe combined immunodeficient mice, and radiolabeled Abs were injected at various times after tumor inoculation. The maximum tolerated dose (MTD), as well as lower doses, was tested. Tumor growth was monitored by hind-leg paralysis. With a 3-5-day interval before Ab injection, anti-CD74 conjugated to either (111)In or (67)Ga, at a dose of 240-350 microCi/mouse, produced a strong therapeutic effect, with greatly delayed tumor growth, and many of the treated mice were tumor free for >6 months. Control mice became paralyzed in 16-24 days, uniformly. Treatment at later time points (9-day interval) had little therapeutic effect. The MTD was required for optimal therapy. With the beta-particle emitter (90)Y, the MTD was much less, 25 microCi/mouse, and at this dose there was only a weak therapeutic effect. In conclusion, the data suggest that low-energy electrons are more effective than beta-particles in this model system. These results may be applicable to humans, particularly in the case of micrometastatic disease. This approach may also be effective with other Abs that accrete in large amounts.     

Radioimmunotherapy of human colonic cancer xenografts with 90Y labeled monoclonal antibodies to carcinoembryonic antigen

Monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA) or alpha-fetoprotein (AFP) were conjugated with diethylenetriaminepentaacetic acid and radiolabeled with 90Y at a specific activity of 4.0-6.0 mCi/mg. Approximately 50% of the radiolabeled anti-CEA antibody (90Y-labeled NP-2) bound to an immunoadsorbent containing CEA while analysis by high performance liquid chromatography revealed that 95-98% of the 90Y was associated with immunoglobulin. Less than 5% of the 90Y dissociated from either MAb after incubation in plasma for 48 h at 37 degrees C. After injection into nude mice, 98% of the circulating radioactivity remained associated with antibody and no loss of immunoreactivity was observed at 3 days. To evaluate 90Y-labeled NP-2 as a therapeutic agent, varied doses (10-100 microCi) were administered as a single i.v. injection into groups of nude mice bearing s.c. implants (0.3-0.4 g) of a CEA-producing human colonic cancer xenograft, GW-39. At the 10-microCi dose, no inhibition of tumor growth was observed. After 28 days, tumor growth was inhibited by as much as 77% in mice treated with 50 microCi of 90Y-labeled NP-2 as compared to tumor growth in control animals given 90Y-labeled anti-AFP. Doses higher than 50 microCi (75 and 100 microCi) were toxic to most of the animals, killing them within 2-3 weeks after administration. Marked suppression of circulating leukocytes was observed with 20 and 50 microCi by 1-2 weeks postinjection, but they returned to normal levels 3-4 weeks later. These studies show that treatment with 90Y-labeled MAbs against CEA can produce significant antitumor effects. However, toxicity to the bone marrow may limit the therapeutic efficacy of systemically administered 90Y-labeled MAbs.     

Effect of mass of 111In-benzyl-EDTA monoclonal antibody on hepatic uptake and processing in mice

In patients, the pharmacokinetic behavior of murine monoclonal antibodies has been observed to vary with the amount of antibody administered. It has been suggested that this reflects human recognition of the foreign mouse protein. We have found that the amount of antibody administered also influenced pharmacokinetic behavior when murine monoclonal antibody was administered to mice. p-Isothiocyanatobenzyl-EDTA, a new chelator which forms complexes with 111In that are stable in vivo, was conjugated to Lym-1, a murine anti-Burkitt's lymphoma monoclonal antibody. The pharmacokinetics of two doses (20 and 0.2 micrograms) of the 111In labeled radiopharmaceutical were studied in non-tumor bearing BALB/c mice. About 20% (0.04 microgram) of the 0.2-microgram dose, compared with 8% (1.6 micrograms) of the 20-micrograms dose, was found in the liver at 48 h after injection. Both doses demonstrated a biological half-life of approximately 120 h. At least 75% of the 111In was excreted by the kidneys, and essentially all 111In in the urine remained chelated by the EDTA portion of p-isothiocyanatobenzyl-EDTA. From these observations of a dose dependent uptake of this radiopharmaceutical by the liver we conclude that there is a recognition phenomenon in mice for this murine monoclonal antibody.     

Hyperthermia enhances localization of 111In-labeled hapten to bifunctional antibody in human colon tumor xenografts

A unique bifunctional antibody (BFA) delivery system was examined for radiolocalization and distribution following hyperthermia (41.5 degrees C, 45 min) of T380h human colon tumor xenografts. The BFA is an F(ab')2 fragment made by combining two murine monoclonal antibodies with different specificities, one directed against carcinoembryonic antigen (monoclonal antibody CEM 231) and the other (monoclonal antibody CHA 255) against a hapten found on a derivative of 111In-labeled benzyl-EDTA (EOTUBE). This BFA is known as CEM/CHA. The CEM/CHA accumulates in carcinoembryonic antigen-expressing tissue and clears from normal tissues prior to administration of the radiolabeled hapten. T380h tumor chunks were injected s.c. into 31 nude mice. Two weeks later mean tumor volume was 352 mm3 and the animals were assigned to one of four groups: (a) CEM/CHA + hyperthermia + 111In-EOTUBE; (b) CHA 255 F(ab')2 + hyperthermia + 111In-EOTUBE, and (c and d) treated in the same manner as a and b, respectively, but without heat. The CEM/CHA, CHA 255 F(ab')2, and 111In-labeled hapten were injected i.p. at 14 micrograms, 7 micrograms, and 140-200 microCi/mouse, respectively. The hyperthermia was administered 22-24 h after BFA and the radiolabeled hapten was injected 2 h later. Twenty-four h thereafter, the animals were euthanized for testing. A significantly greater percentage of injected radioactivity localized within heated compared to unheated tumors in mice given CEM/CHA and 111In-EOTUBE (7.39%/g tumor and 4.46%/tumor versus 2.72%/g tumor and 1.44%/tumor, respectively). The percentage of kidney activity in mice given CHA 255 F(ab')2 fragments and heat was 57% lower than in the nonheated group when expressed on a per g basis (12.73 and 22.20%, respectively). Microautoradiography showed greater radiolocalization in heated tumors than in nonheated control tumors of comparable size. Semiquantification by immunoperoxidase staining for carcinoembryonic antigen did not reveal similar differences in the amounts of antigen present in tumors from heated and nonheated groups. These findings suggest that hyperthermia could be used to enhance delivery of radiolabeled haptens to prelocalized BFA and, thus, to enhance tumor imaging and therapeutic efficacy.     

Whole-body autoradiography of tumor-bearing hamsters with a new tumor imaging agent, indium-111-labeled porphyrin

We have synthesized a new tumor imaging agent, 111In-labeled porphyrin (111In-ATN-2). In order to image transplantable pancreatic carcinoma in Syrian golden hamsters, we investigated the biodistribution of 111In-ATN-2 72 hr after injecting the agent by means of whole-body autoradiography. The efficacy of the agent was compared with that of 67Ga citrate. The images with 111In-ATN-2 were found to be clearer than those with 67Ga citrate. Tumor-to-tissue radiodistribution ratios of the former were higher than those of the latter. Thus, 111In-ATN-2 seems to be more useful for tumor diagnosis.     

Enhanced killing of human small cell lung cancer by hyperthermia and indium-111-bleomycin complex.

Indium-111-bleomycin complex (111In-BLMC) is a radiopharmaceutical agent that produces tumor regression in mouse glioma in vivo and kills human small cell lung cancer (SCLC) cells in vitro. The interaction between hyperthermia and 111In-BLMC against human SCLC (N417) cells was studied for bleomycin (BLM) (15 micrograms/ml) or 111In-BLMC (40-50 microCi carried by 15 micrograms BLM/ml) for 5 min or 1.5, 2, or 4 hr at 37 degrees C or 43 degrees C exposures. Cell survival was determined by colony formation in soft agarose. There was a synergistic effect for 111In-BLMC and hyperthermia for cell killing. At 37 degrees C, the percent survival of N417 cells for BLM alone was 25.9%, and for 111In-BLMC it was 13.2%; at 43 degrees C, survival was 5.3% for BLM alone and 1.2% for 111In-BLMC by a 4 hr treatment. Effectiveness was greater when 111In-BLMC was combined with hyperthermia.     

Effect of interleukin-2 on biodistribution of monoclonal antibody in tumor and normal tissues in mice bearing SL-2 thymoma.

BACKGROUND: Our laboratory demonstrated previously that treatment with a tumor-specific, radiolabeled anti-Thy 1 murine monoclonal antibody (MAb) in mice can eradicate a T-cell lymphoma mass, but the doses required were toxic to normal organs. Approaches to increase MAb concentration in tumor tissue versus normal tissue may overcome this problem. Interleukin-2 (IL-2) has been shown to increase capillary permeability, as indicated by extravasation of albumin. PURPOSE: The purpose of this study was to determine whether increased extravasation of MAb at the tumor site might result in selective binding to tumor antigen, increasing localization of radiolabeled MAb at the tumor site. METHODS: We studied the effect of IL-2 on biodistribution of 1A14 MAb (anti-Thy 1.1) in normal AKR/Cum (Thy 1.2+) mice and in AKR/Cum mice bearing SL-2, a spontaneous T-cell lymphoma (Thy 1.1+), compared with biodistribution of albumin in normal mice and biodistribution of the nonreactive G3G6 MAb in tumor-bearing mice. IL-2 was given intravenously in the tail vein in doses of 0, 25,000, 50,000, 100,000, or 200,000 U twice a day for a total of seven doses over 3.5 days. Mice received injections of a mixture of 1A14 MAb (250 muCi/100 micrograms) and albumin or G3G6 MAb (145 muCi/100 micrograms) in a total volume of 200 microL at 12 hours after the last IL-2 dose. RESULTS: In normal mice, IL-2 caused a dose-dependent increase of both radiolabeled MAb and albumin in the spleen, liver, lung, and lymph node, but it spared the brain. In tumor-bearing mice, IL-2 resulted in higher levels of MAb in the tumors 72 hours after receiving injections, with 17.5% and 24.3% of the injected dose per gram of tumor present in the mice pretreated with 100,000 or 200,000 U of IL-2 twice a day for 3.5 days, compared with 13.4% in the controls. In IL-2-treated mice, levels of MAb were greater in the tumors than in critical normal organs; the differences were statistically significant for tumors versus lungs at 24 hours after injection and for tumors versus livers at 48 hours and 72 hours after injection. CONCLUSIONS: These results suggest that pretreatment with IL-2 may lead to enhanced distribution of tumor-specific MAb to the tumor site, compared with normal tissues, thus increasing therapeutic efficacy of radiolabeled MAb.