Treatment of lymphoma with radiolabeled antibody: elimination of tumor cells lacking target antigen

We have previously shown that 131I-labeled monoclonal antibodies against the Thy1.1 differentiation antigen can induce the regression of Thy1.1 antigen-positive (Thy1.1+) AKR/J SL2 T cell lymphoma nodules in AKR/Cum (Thy1.2+) mice. In this study, we examined the ability of 131I-labeled anti-Thy1.1 antibodies to eliminate tumor nodules containing variant lymphoma cells that do not express the Thy1.1 antigen (Thy1.1-). AKR/Cum mice were sc inoculated with mixtures of 1-2 X 10(7) AKR/J SL2 cells and varying amounts (0.3%-10%) of Lsp3, a Thy1.1 antigen-negative (Thy1.1-) subclone of the SL2. One week later, when an established tumor nodule was present, mice were treated with 1,500-1,700 microCi of 131I-labeled anti-Thy1.1 antibody. Complete regression of tumor was observed in 11 of 12 (92%) mice inoculated with tumor containing 0.3%-1% antigen-negative cells. In contrast, no complete regressions were observed in mice with only antigen-negative tumor cells treated with 131I-labeled anti-Thy1.1 antibody or in mice inoculated with antigen-positive tumor and treated with an 131I-labeled control antibody. Of the mice receiving mixtures containing 3%-10% antigen-negative cells, five of seven showed complete regression. These results demonstrate that radiolabeled antibodies can eliminate small numbers of antigen-negative tumor cells present within a tumor mass.     

Biodistribution and dosimetry following infusion of antibodies labeled with large amounts of 131I.

Dosimetry and treatment planning for therapeutic infusions of radiolabeled antibodies are usually performed by extrapolation from the biodistribution of trace-labeled antibody. This extrapolation assumes that the biodistribution of high specific activity antibody will be similar to that seen with trace-labeled antibody. However, high doses of radiation result in rapid depletion of lymphoid and hematopoietic cells in lymph nodes, spleen, and marrow with replacement by blood and plasma. If radiolabeled antibody is cleared slowly from blood, this replacement may result in increased radionuclide concentrations in these tissues following infusions of antibody labeled with large amounts of radionuclide. To examine the influence of deposited radiation on the biodistribution of radiolabeled antibody, we treated mice with a constant amount of antibody that was labeled with varying amounts of 131I. Survival was determined in normal specific pathogen-free AKR/Cum mice (Thy1.2+) after infusion of anti-Thy1.1 antibody labeled with 10 to 6500 muCi of 131I, to determine an appropriate range of 131I doses for further study. The dose producing 50% lethality within 30 days following infusion of 131I-labeled antibody was 530 muCi 131I. Biodistribution, bone marrow histology, and dosimetry were subsequently determined after infusion of 500 micrograms of antibody labeled with 10, 250, 500, or 3500 muCi 131I. The amount of 131I did not influence uptake or retention of antibody in blood, liver, lung, or kidney. In contrast, infusion of antibody labeled with 250 to 3500 muCi of 131I led to a dose-related increase in the concentration of 131I in marrow, spleen, lymph node, and thymus. For example, at 96 h after infusion of antibody labeled with 500 or 3500 muCi 131I, concentrations in marrow were 3- to 4-fold higher than after infusion of trace-labeled antibody. The increase in marrow 131I concentrations was associated with depletion of cells and hemorrhage within the marrow space. As a result, estimated mean absorbed doses to marrow, lymph node, spleen, and thymus were 1.2 to 3.1 times higher than would have been predicted from the biodistribution of trace-labeled antibody. These results suggest that the biodistribution of trace-labeled antibody should be an accurate predictor of the behavior of high specific activity antibody in blood and solid organs such as liver and kidney. In contrast, radiation from antibody labeled with large amounts of radionuclide can result in an alteration of the concentration of radiolabeled antibody in rapidly responding tissues such as marrow.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental radiotherapy of murine lymphoma with 131I-labeled anti-Thy 1.1 monoclonal antibody

Monoclonal antibodies against the Thy 1.1 differentiation antigen are ineffective in the treatment of transplanted AKR T-cell lymphoma once a palpable tumor nodule is present, due to the inability of the host to eliminate antibody-coated tumor cells. To overcome this limitation, we have evaluated the use of 131I-labeled anti-Thy 1.1 antibodies for the therapy of established AKR/J SL2 lymphoma (Thy 1.1+) nodules growing in congeneic AKR/Cu mice (Thy 1.2+). In these experiments, 131I-anti-Thy 1.1 antibody specifically localized to a s.c. tumor with a mean of 6.5% of the infused dose per g of tumor at 24 h after infusion. The proportion of infused anti-Thy 1.1 antibody localizing to tumor was constant following antibody doses of up to 400 micrograms/animal. Antibody iodinated with up to 2 atoms of iodine per antibody of molecule maintained binding activity and localization to tumor equivalent to antibody labeled with less iodine. The concentrations of 131I-anti-Thy 1.1 in tumor would result in delivery of a mean of 1600 cGy to tumor following infusion of 500 muCi of 131I-labeled anti-Thy 1.1 antibody. In comparison, 500 muCi 131I-labeled irrelevant antibody would deliver a mean of 380 cGy to tumor. Treatment of animals with palpable tumor nodules with 500 muCi 131I-anti-Thy 1.1 led to regression of the tumor nodule in 44% of animals, significantly prolonged survival, and cured two of five of the animals treated prior to the development of metastatic disease. In contrast, unlabeled anti-Thy 1.1 led to tumor response in 6% of animals, and up to 1000 muCi 131I-labeled irrelevant antibody had no effect on tumor growth. Therapy was limited by the emergence of variant tumor cells lacking the target antigen and by bone marrow toxicity following 131I-labeled antibody doses of greater than or equal to 1000 muCi/animal. These studies demonstrate that 131I-labeled monoclonal antibodies can have a significant antitumor effect in a situation where unmodified antibody is ineffective.     

Enhancement of monoclonal antibody binding to melanoma with single dose radiation or hyperthermia

We undertook this study to determine whether radiation (10 Gray, single dose) or water bath hyperthermia (41 degrees C, 45 min) could enhance binding of 111In-labeled anti-p97a monoclonal antibody (MAb) to human melanoma tumors transplanted subcutaneously into nude mice. Sixty animals were given injections of 1-2 X 10(7) Brown C5513 melanoma cells. At 1-2 weeks postinjection, two-thirds of the mice were treated (one-third served as controls). Within 3 hours after treatment, each animal was given iv 2 muCi 111In-anti-p97a MAb. At 24 and 48 hours thereafter, whole-body scans were done with the use of a MaxiCamera 300 A/M unit, and the ratio of activity at the tumor and liver was determined. Some animals were kept for 7 days posttreatment, whereas others were taken after the 48-hour scan for determination of biodistribution of the radiolabeled complex. Enhancement of MAb binding was demonstrated by either modality, although enhancement was more consistent with radiation. The therapeutic efficacy of MAb may be enhanced with increased binding of radioactive MAb complexes through single dose radiation or hyperthermia.     

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.     

Monoclonal antibody therapy of murine lymphoma: enhanced efficacy by concurrent administration of interleukin 2 or lymphokine-activated killer cells

Lymphokine-activated killer (LAK) cells have recently been shown to be very efficient effector cells for antibody-dependent cellular cytotoxicity. Thus, we explored, in a murine lymphoma model, administration of LAK-inducing doses of interleukin 2 (IL-2) or adoptive transfer of LAK cells as a means of enhancing therapy with tumor-specific monoclonal antibody (mAb). AKR/Cum (Thy-1.2+) hosts were inoculated on day 1 s.c. with the SL-2 thymoma of AKR/J origin (Thy-1.1+) and developed palpable tumor on day 4. Tumor-specific anti-Thy-1.1 IgG2a mAb, 1A14, was given on days 4 and 8 with 50,000 units/day IL-2 i.p. divided in two doses on days 4-12. Therapy with IL-2 or mAb alone had minimal activity, prolonging control median survival of 22 days to 25 and 29 days, respectively, whereas therapy with IL-2 plus mAb significantly prolonged median survival to 40 days. However, combined therapy did not result in cures and long term survival. The efficacy of combined therapy did not result from alterations in the biodistribution of mAb by concurrent IL-2 infusions, as determined by studies with radiolabeled mAb. The combined effect of in vitro generated LAK (10(8) cells) adoptively transferred i.v. with 1A14 on days 4 and 8 following SL-2 inoculation was also evaluated. This regimen had no detectable toxicity, and treatment of mice with LAK and mAb resulted in 60% long term survival compared with 17% or 0% for mice treated with mAb or LAK alone. Thus, the therapeutic effects of tumor-specific mAb was enhanced by in vivo administration of IL-2 or by adoptively transferred LAK, which may represent means to provide the host with increased antibody-dependent cellular cytotoxicity effector cells. Adoptively transferred LAK has the additional benefit of augmenting mAb therapy of tumor without the toxicity associated with the induction of such cells in vivo with high dose IL-2.     

Comparative localization of murine monoclonal antibody Me1-14 F(ab')2 fragment and whole IgG2a in human glioma xenografts

Monoclonal antibodies (MAbs) targeted to glioma-associated antigens may allow the selective delivery of imaging and therapeutic agents to brain tumors; the use of MAb fragments may be a strategy to further improve tumor uptake of such agents relative to normal tissues. In this study, we have examined the in vivo localization of radioiodinated MAb Me1-14, a murine immunoglobulin G2a (IgG2a) reactive with gliomas, and its F(ab')2 fragment in s.c. and intracranial xenografts of human glioma cell line D-54 MG in athymic mice. The radiolabeled F(ab')2 fragment of Me1-14 was demonstrated to possess in vitro binding affinity and immunoreactivity comparable to that of whole IgG. Direct comparison of IgG and F(ab')2 biodistribution in s.c. xenograft-bearing mice showed higher tumor: normal tissue ratios of the F(ab')2 fragment compared to IgG. In intracranial tumor-bearing mice paired-label analysis using a nonspecific protein control showed earlier specific tumor localization by the F(ab')2 fragment of Me1-14 compared to IgG. Blood-to-tumor transfer constants (K1) derived for Me1-14 F(ab')2 were significantly greater than those for whole Me1-14 (P = 0.01). Estimated radiation dosimetry revealed that 131I-labeled Me1-14 F(ab')2 would deliver higher radiation doses to tumor than to normal tissues. These studies demonstrate that the F(ab')2 fragment of Me1-14 may be a potential agent for immune-directed brain tumor diagnosis and therapy.     

Advantage of dose fractionation in monoclonal antibody-targeted radioimmunotherapy

Monoclonal antibody (MAb) B72.3 IgG was radiolabeled with 131I and administered to female athymic NCr-nu mice bearing the LS-174T human colon adenocarcinoma xenograft to determine if fractionation of MAb dose had any advantage in tumor therapy. In the LS-174T xenograft, only approximately 30%-60% of tumor cells express the B72.3-reactive TAG-72 antigen. The LS-174T xenograft was used to reflect the heterogeneity of the TAG-72 antigen often seen in biopsy specimens from patients. In contrast to a single 600-muCi dose of 131I-B72.3 IgG where 60% of the animals died from toxic effects, two 300-muCi doses of 131I-B72.3 IgG (total of 600 muCi) reduced or eliminated tumor growth in 90% of mice, with only 10% of the animals dying from toxic effects. Dose fractionation even permitted escalation of the dose to three doses (each 1 wk apart) of 300 muCi of 131I-B72.3 IgG (for a total of 900 muCi), resulting in even more extensive tumor reduction or elimination and minimal toxic effects. The use of an isotype-matched control MAb revealed a nonspecific component to tumor growth retardation, but the use of the specific B72.3 IgG demonstrated a much greater therapeutic effect. Tumors that had escaped MAb therapy were analyzed for expression of the B72.3-reactive TAG-72 antigen with the use of the immunoperoxidase method; they were shown to have the same antigenic phenotype as the untreated tumors. We verified tumor elimination by killing the test animals after a 7-week observation period and performing histologic examination of tumor sites. We also monitored toxic effects by histologic examination of numerous organs, including bone marrow. These studies thus demonstrate the advantage of dose fractionation of a radiolabeled MAb for tumor therapy. We anticipate that the concept of dose fractionation can be practically applied in radioimmunotherapeutic clinical trials with the development and use of recombinant-chimeric MAbs and modified constructs.     

Tumor targeting properties of indium-111 labeled genetically engineered Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment (chTNT)-3 antibody

Genetic engineering techniques have allowed the construction of Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment antibody (chTNT-3), a chimeric monoclonal antibody (MAb) that targets necrotic regions of solid tumors. The purpose of this study is to evaluate the in vitro and in vivo properties of Fab' and F(ab')2 constructs radiolabeled with indium-111 (111In) using diethylentriamine pentaacetic acid (DTPA) conjugation to develop a clinically useful imaging agent for the detection of necrosis in solid tumors. Optimization of the MAb-to-DTPA ratio showed that a 1:2 ratio gave the best immunoreactivity while providing good radiolabeling efficiency and high specific activity for all three DPTA conjugates. In addition, 111In-labeled Fab' and F(ab')2 conjugates were found to have faster whole body clearance times and better biodistribution profiles compared to parental 111In-labeled chTNT-3 in tumor-bearing mice. Although radiolabeled Fab' and F(ab')2 constructs showed lower tumor uptake than radiolabeled chTNT-3, biodistribution results showed that these constructs had significantly lower uptake in liver, spleen, and other normal organs (except the kidney), and therefore had higher tumor-to-organ ratios. In addition, a comparison of all derivatives showed that the F(ab')2 reagent gave the best results in tumor imaging studies. These results demonstrate that stable, a genetically engineered F(ab')2 construct can be successfully radiolabeled with 111In to produce potential imaging reagents for the imaging and monitoring of tumor necrosis.     

Monoclonal antibody therapy of spontaneous AKR T-cell leukemia

We have previously shown that monoclonal antibodies against the Thy 1.1 differentiation antigen can inhibit the outgrowth of a lethal inoculum of transplanted AKR T-leukemic cells. In the present report we have extended these studies to examine antibody therapy of aged AKR/J mice with spontaneous leukemia. Infusion of anti-Thy 1.1 antibody in frankly leukemic mice led to uniform early mortality from cell lysis and agglutination. In contrast, anti-Thy 1.1 antibody therapy of mice in remission following treatment with cyclophosphamide prolonged remission duration (P less than 0.001) and modestly prolonged survival (P less than 0.01) compared to treatment with irrelevant antibody or chemotherapy alone. The major cause of failure was relapse of leukemia. In 85% (47 of 55) of cases relapse was due to cells that continued to express Thy 1.1, but in 15% of these relapsing animals all leukemic cells failed to express the target antigen. Our results suggest that monoclonal antibody against a normal T-cell antigen can add to the antileukemic effects obtained with chemotherapy alone. Nevertheless, the clinical benefit of unmodified antibody was modest, and antibodies conjugated to cytotoxic agents may be needed to overcome the limitations of unmodified antibodies.     

Breast-tumor xenograft targeting and therapy studies using radiolabeled chimeric anti-cea monoclonal-antibody t84.66

Carcinoembryonic antigen (CEA) is expressed in approximately 60% of breast carcinomas. T84.66 is a well characterized anti-CEA murine monoclonal antibody (MAb) that does not cross-react with other CEA-related proteins. It has been humanized and used extensively after radiolabeling in clinical/experimental protocols for localization/therapy of colorectal cancer. MCF-7 human breast cancer cell line expresses CEA and is tumorigenic in athymic mice. In order to determine if anti-CEA MAb could specifically target breast cancer CEA, biodistribution, radiolocalization and therapy studies were performed in the animal model. Five tumor-bearing animals per time point were injected with 15 muCi of In-111-cT84.66. Mice were imaged and sacrificed at 24, 48, 72 and 144 h and biodistribution studies performed. For therapy studies, 19 tumor-bearing mice were injected either with 120 muCi of 90Y-cT84.66 or PBS. The tumors were measured tri-weekly and weighed at autopsy 21 days post therapy. Activity accumulation steadily increased in the tumors reaching 21% of the injected dose per gram of tumor (ID/g) at 144 h. At that time, the %ID/g in the tumor was 3 times higher than that in blood and the liver and 8 times higher than in other major organs. On day 10 post-therapy, 16 of the 19 control mice had tumor volumes between 1 and 3 cm3, while none of the treated tumors ever reached 1 cm3 in size. At autopsy, a 12-fold tumor weight difference (p=0.0001) was observed between control and treated mice (2.4g vs. 0.2g average weight, respectively). In summary, breast cancer CEA was specifically targeted with T84.66 allowing good tumor localization as well as significant tumor growth inhibition. Given the significant expression of CEA in breast cancer, this tumor should be included into the CEA-expressing malignancies for targeting with anti-CEA MAbs.     

Influence of dose and schedule on the therapeutic efficacy of 131I-labelled monoclonal antibody 139H2 in a human ovarian cancer xenograft model.

The therapeutic efficacy of various doses and schedules of 131I-labelled anti-episialin monoclonal antibody (MAb) 139H2 was assessed in the NIH:OVCAR-3 human ovarian cancer xenograft model. Radioimmunotherapy was started at the time s.c. tumors were well established (100 to 300 mm3). The anti-tumor effects induced by i.v. injections of 131I-MAb 139H2 were dose- and schedule-dependent. Optimal growth inhibition and long-lasting complete tumor regressions were obtained with 2 injections of 500, 700 or 750 muCi 131I-MAb 139H2 per mouse given with a 2-week interval. The percentage of tumors with more than 50% reduction of their initial volume after treatment with a total dose of 1,000 muCi 131I-MAb 139H2 per mouse, given as 10 injections of 100 muCi (3 x/week), 4 injections of 250 muCi (2 x/week), 10 injections of 100 muCi (5 x/week) within a period of 3 weeks, or 2 injections of 500 muCi with a 2-week interval, was 9%, 40%, 64% and 75% respectively. Unlabelled MAb 139H2 did not affect tumor growth, while the effects of 131I-control MAb were minor and transient. 131I-MAb 139H2 treatment did not select for outgrowth of episialin-negative cells in the OVCAR-3 xenografts. Highest absorbed doses of whole-body-radiation were calculated for 2 injections (500 to 750 muCi 131I-MAb 139H2) given with the 2-week interval. The radiation dose to the tumor after a single injection of 500 muCi 131I-MAb 139H2 was 1,300 cGy over 7 days, which appeared slightly lower than the dose calculated after administration of a tracer dose of iodinated MAb 139H2.     

Enhanced tumor uptake of macromolecules induced by a novel vasoactive interleukin 2 immunoconjugate

Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive drugs or peptides linked to tumor-specific MAbs. To test this hypothesis, two MAbs, Lym-1 directed against human malignant lymphomas and B72.3 reactive with the TAG-72 antigen expressed in solid tumors, were chemically conjugated with human recombinant interleukin 2 (IL-2). IL-2 has been used in humans to activate lymphokine-activated killer cells for the treatment of cancer but is also known to produce a generalized vascular permeability by an unknown mechanism when used systemically. Chemical conjugation of IL-2 to MAbs appears to destroy its cytokine function as shown by T-cell proliferation studies in vitro. Despite this finding, MAb/IL-2 immunoconjugates retain their ability to produce an enhanced vascular permeability when injected i.v. into nude mice bearing relevant tumor models only. Biodistribution studies using 125I-labeled tracer Lym-1 have demonstrated that the Lym-1/IL-2 immunoconjugate can increase antibody uptake in tumor by a factor of 4 in a time (2.5-h pretreatment)- and dose (30 micrograms/mouse)-dependent manner. In contrast, treatment of mice with free IL-2 and antibody showed this effect in all organs of the mouse including the tumor. Bidirectional crossover imaging studies in individual tumor-bearing nude mice showed improved uptake and decreased blood pool when the MAb/IL-2 immunoconjugates were used compared to controls. Finally, tumor blood flow and vascular permeability studies demonstrate that the physiological effect of the MAb/IL-2 is due to a reversible and specific vascular leakage at the tumor site. These studies indicate that pretreatment with this novel immunoconjugate may enhance the diagnostic and therapeutic potential of MAbs, drugs, and other macromolecules for the treatment of cancer.     

Characterization of in vivo suppression of syngenic tumor by allogenic effector cells

The purpose of this study was to characterize the effects of allogenic splenocytes transferred with tumor cells, subcutaneously, to a host syngenic for tumor cells. Cytotoxic effector cells usually resulted in tumors less than half the size of controls at E:T of 10:1. Primary immune splenocytes were more effective than hyperimmune splenocytes in delaying tumor growth. Actively cytotoxic splenocytes by in vitro ⁵¹Cr release assay were required for delayed tumor growth; memory cell populations were not effective. Delayed tumor growth correlated with in vitro cytotoxicity of primary immune splenocytes; however, hyperimmune splenocytes, even though they possessed greater in vitro cytotoxic responses, showed lesser tumor suppression in vivo. T cells were necessary for tumor suppresion, as treatment of B6AF1 effector cells with anti-Thy 1.2 serum abrogated suppression; T cell enrichement by nylon-wool treatment of effector cells increased tumor suppression. Delay in tumor growth was an in vivo phenomenon, for anti-Thy 1.2 serum in AKR hosts abrogated the effect of Thy 1.2 effector cells.     

Radioimmunotherapy for Pancreatic Carcinoma Using 131I-Labeled Monoclonal Antibody Nd2 in Xenografted Nude Mice

We investigated the biodistribution, radiolocalization, and radioimmunotherapeutic potential of ¹³¹I-labeled Nd2 in athymic nude mice bearing human pancreatic carcinoma xenografts. ¹³¹I-Nd2 was accumulated at high levels in the tumor, in contrast to blood, liver, spleen, and other normal organs. The tumor was clearly delineated in scintigraphs. The volumes of tumors of mice injected with 7.4 MBq of ¹³¹I-Nd2 were 80% less than those of tumors before injection of radiolabeled Nd2. Fibrous or vacuolar degeneration was seen in histological sections of tumors of 7-week-treated mice. The growth of tumors in mice treated with misonidazole, a hypoxic cell radiosensitizer, and then injected twice with 3.7 MBq of ¹³¹I-Nd2 was suppressed over 7 weeks. Neither leucocytopenia nor thrombocytopenia was severe after injection of radiolabeled Nd2. Thus ¹³¹I-labeled Nd2 may have clinical application in the radioimmunotherapy of pancreatic cancer.     

Enhanced Tumor Localization of Radiolabeled Fab Fragments of Monoclonal Antibody A7 in Nude Mice Bearing Human Pancreatic Carcinoma Xenografts

Much recent research has been directed toward the use of monoclonal antibodies (MAb) for the inimunodetection of solid tumors. In pancreatic cancer, the results of conventional immunoscintigraphy using intact MAb remain disappointing. Clear immunoseintigrapliy with radiolabeled MAb requires a high tumor tissue/blood ratio of radioactivity and a low normal tissue/blood ratio of radioactivity. In this study, ¹²⁵I-labeled Fab fragments produced by papain digestion of MAb A7 were injected intravenously into nude mice bearing a human pancreatic cancer (HPC-YS) xenograft previously shown to react specifically with MAb A7. The radioactivity of tumors and normal organs was subsequently measured. The tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was 1.00±0.24 and 9.68±2.54 at 2 and 24 h after injection, respectively. The tumor tissue/blood ratio of radioactivity was significantly higher than those of normal organs at 24 h after injection. Moreover, the tumor tissue/blood ratio of ¹²⁵I-labeled Fab fragments of MAb A7 was greater than that of intact MAb A7, although the ¹²⁵I-labeled Fab accumulation level was much less than that of ¹²⁵I-labeled intact MAb A7 in the tumor. When mice bearing tumors which did not react with MAb A7 were studied, ¹²⁵I-labeled Fab fragments did not specifically localize to the tumors. These results suggest that Fab fragments of MAb A7 may be suitable carriers of radionuclides for the immunodetection of human pancreatic cancer.     

The presence of a concomitant bulky tumor can decrease the uptake and therapeutic efficacy of radiolabeled antibodies in small tumors.

We describe the effects that a concomitant large tumor mass can exert on the therapeutic efficacy of radioimmunotherapy against small tumors, using the nude mouse/GW-39 human colonic cancer model. The tumor uptake 7 days p.i. of i.v.-injected 131I-labeled anti-CEA MAb (NP-4) and anti-CSAp MAb (Mu-9) in small (less than 0.2 g) s.c. GW-39 tumors was approximately 3-fold lower in animals with a concomitant large (greater than 1.0 g) GW-39 tumor than in the absence of a large tumor. An inverse correlation between the mass of the tumor burden and the 131I levels in the blood was observed, indicating that a large tumor mass may act as a sink for the injected radiolabeled antibody. Increasing antibody protein dose did not reverse the reduced uptake in the small s.c. tumors. The therapeutic efficacy of a single 0.25-mCi injection of 131I-labeled anti-CSAp MAb Mu-9 towards intrapulmonary GW-39 metastases was tested in nude mice bearing either small or large GW-39 s.c. tumors. Over 80% of the animals with small s.c. GW-39 tumors survived 18 weeks after tumor transplantation, whereas less than 20% of the animals bearing large s.c. tumors survived past 13 weeks. Dosimetric calculations, based on biodistribution data over time, indicated that the presence of a large s.c. tumor mass may have decreased the radiation dose to the intrapulmonary tumors almost two-fold. However, the radiation dose to the blood was also decreased in the animals with the large tumor burden. Therefore, the animals with larger tumor burden may also have been able to sustain higher doses of the radioantibody. The presence of a large tumor mass can thus affect the biodistribution and therapeutic efficacy of radioiodinated antibodies. We suggest that bulky tumors can adsorb a considerable amount of the injected dose, thereby reducing the total amount of MAb available for binding to the smaller tumors.     

Induction of antitumor immunity by combined immunogene therapy using IL-2 and IL-12 in low antigenic Lewis lung carcinoma

Interleukin-2 (IL-2) and interleukin-12 (IL-12) are crucial cytokines that induce potent antitumor responses in a variety of animal cancer models. Although single gene transfer of either IL-2 or IL-12 exhibits limited antitumor effects, the combination of IL-2 and IL-12 has been shown to induce a stronger antitumor response and to cure tumor-bearing mice. To examine the conditions necessary for tumor rejection, we varied the local concentration of IL-2 and IL-12 by introducing these genes into Lewis lung carcinoma (LLC) cells via retroviral vectors and/or an adenoviral vector and evaluated the growth of inoculated LLC cells (5 x 10(5) cells). In contrast to the result when using a stepwise dose increase of IL-2 either without or with a fixed production of IL-12 (4-5 ng/5 x 10(5) cells/24 hours, insufficient for tumor rejection by itself), rejection of the tumor was achieved in 75% of the mice when the IL-12 secretion was combined with high and transient IL-2 production (42 ng/5 x 10(5) cells/24 hours) using additional adenoviral vector transduction (100 multiplicities of infection). An abundant infiltration of both CD4+ (47.4/mm2) and CD8+ (85.6/mm2) T cells was a characteristic finding in the dual gene-transfected LLC tumors. Importantly, consistent with the rejection of rechallenged parental cells, tumor-specific cytotoxic T lymphocytes were induced only from the splenocytes of mice inoculated with the dual gene-transduced LLC cells, suggesting the existence of protective antitumor memory. In addition, only vaccination of dual gene-transduced LLC cells inhibited the growth of pre-established LLC tumors. These results indicate that generation of a pivotal antitumor response likely depends on the synergistic combination and concentration of IL-2 and IL-12 in the local milieu by which tumor-specific immune memory is established.     

111Indium‐labeled monoclonal antibody K1: Biodistribution study in nude mice bearing a human carcinoma xenograft expressing mesothelin

The monoclonal antibody (MAb) K1 is a murine IgG₁ that recognizes mesothelin, a differentiation antigen present on mesothelium which is highly expressed on cancers derived from mesothelium, including most ovarian cancers and epithelioid mesotheliomas. MAb K1 was conjugated to 2‐(p‐isothiocyanatobenzyl)‐cyclohexyl‐ diethylenetriaminepentaacetic acid and labeled with ¹¹¹In. The biodistribution of ¹¹¹In‐K1 was studied in athymic nude mice bearing 2 s.c. tumors, one expressing a stably transfected plasmid encoding mesothelin and one composed of the parental untransfected A431 epidermoid carcinoma cells which do not express mesothelin. Tumor‐bearing mice were given an i.v. injection of ¹¹¹In‐K1 and killed at different time points to determine the uptake of radiolabeled antibody. Significantly higher uptake was seen in antigen‐positive tumors at all time points, with peak values at 72 hr (52.9% vs. 8% of the injected dose/g tissue for antigen‐positive and antigen‐negative tumors, respectively). Uptake in antigen‐positive tumors was higher than the blood level at all time points, and the tumors contained a high level of the radiolabeled MAb even at 7 days (28.6% of the injected dose/g tumor). Int. J. Cancer80:559–563, 1999. Published 1999 Wiley‐Liss, Inc.     

Expression of Thy 1 antigen in normal and neoplastic mammary cells of mice.

The expression of Thy 1.2 (theta C3H) antigen was measured on the membranes of normal and neoplastic RIII and C3H mammary cells. Competitive inhibition assays revealed that the average membrane content of Thy 1.2 in mammary tissues was about equal to that of lymph node cells. Higher percentages of Thy 1.2-positive cells than mammary tumor virus (MuMTV)-positive cells were observed by immunofluorescence, which suggested that not all the Thy 1.2-positive cells recovered from tumors were also MuMTV-positive. Established tissue culture cell lines C3H and RIII MT expressed lower levels of Thy 1.2 than did cells from mammary tumors. Treatment with the synthetic gluco-corticoid dexamethasone increased the average Thy 1.2 expression in cultured mammary tumor cells as well as the levels of RNA-directed DNA polymerase. Since the percentages of Thy 1.2-positive cells also were greater in steroid-treated cultures, while fewer cells were needed to absorb a standard amount of anti-Thy 1.2 activity, it was concluded that dexamethasone enhanced membrane Thy 1.2 expression as well as MuMTV production by the cultured cells.