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.     

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

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

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

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

Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors.

The uptake and binding of monoclonal antibodies (MAbs) in solid tumors after a bolus i.v. injection are described using a compartmental pharmacokinetic model. The model assumes that MAb permeates into tumor unidirectionally from plasma across capillaries and clears from tumor by interstitial fluid flow and that interstitial antibody-antigen interactions are characterized by the Langmuir isotherm for reversible, saturable binding. Typical values for plasma clearance and tumor capillary permeability of a MAb and for interstitial fluid flow and interstitial volume fraction of a solid tumor were used to simulate the uptake of MAbs at various values of the binding affinity or antigen density for a range of MAb doses. The model indicates that at low doses, an increase in binding affinity may lead to an increase in MAb uptake. On the other hand, at doses approaching saturation of antigen or when uptake is permeation limited, an increase in the binding affinity from moderate to high affinity will have only a small effect on increasing MAb uptake. The model also predicts that an increase in antigen density will greatly increase MAb uptake when uptake is not permeation limited. Our experiments on MAb uptake in melanoma tumors in athymic mice after injection of 20 micrograms MAb (initial plasma concentration, about 120 nM) are consistent with these model-based conclusions. Two MAbs differing in affinity by more than 2 orders of magnitude (3.8 x 10(8) M-1 and 5 x 10(10) M-1) but with similar in vivo antigen densities in M21 melanoma attained similar concentrations in the tumor. Two MAbs of similar affinity but having a 3-fold difference in in vivo antigen density in SK-MEL-2 melanoma showed that the MAb targeted to the more highly expressed antigen attained a higher MAb concentration. We also discuss the model predictions in relation to other experiments reported in the literature. The theoretical and experimental findings suggest that, for high dose applications, efforts to increase MAb uptake in a tumor should emphasize the identification of an abundantly expressed antigen on tumor cells more than the selection of a very high affinity MAb.     

Pharmacokinetics of the mouse monoclonal antibody 17-1A in cancer patients receiving various treatment schedules

Twenty-four patients with metastatic colorectal carcinoma were treated with repeated doses (200-500 mg) of the mouse monoclonal antibody (MAb) 17-1A. Four different treatment schedules were used. The total dose was 1, 3.6, 7.6, and 12 g, respectively. Altogether, 263 infusions were administered. The interindividual variations in the maximum serum concentration at 2 h (max2 h) were large. The mean max2 h value after an infusion of 200 mg was 55 +/- 5 micrograms/ml and after 500 mg, 132 +/- 7 micrograms/ml. Max2 h concentration correlated inversely with the half-life of MAb 17-1A (P less than 0.001). The t1/2 beta for 200 mg was 25.9 +/- 1.4 h and after the administration of 500 mg, 19.8 +/- 1.0 h. The total area under the concentration versus time curve increased when high doses were administered on a continuous basis, in comparison with spaced infusions, thus increasing the exposure of the tumor tissues to MAb 17-1A. The pharmacokinetics of mouse MAb 17-1A are best described by a one-compartment model. All patients developed anti-mouse IgG antibodies and most also IgM antibodies. In the more intensive treatment schedules, the IgG antibody response was suppressed. Induction of high titers of anti-mouse antibodies did not cause clinical problems. Neither did they affect the pharmacokinetics of MAb 17-1A at these dose levels. Therapy was tolerated well. The side effects were mild and of short duration. The gastrointestinal adverse reactions were dose dependent and correlated to serum max2 h concentration. Allergic reactions were rare and easily clinically manageable.     

Significance of circulatory clearance of tumour-localising IgG and F(ab')2 for potential therapy studied in a CEA-producing xenograft model

The significance of circulatory clearance of tumour-localising IgG and F(ab')2 for potential cancer therapy has been studied in immunodeprived mice bearing a carcinoembryonic antigen (CEA)-producing colon tumour. Intact radiolabelled anti-CEA (1H12) exhibited a prolonged localisation in tumour up to 8 days with injected doses between 4 and 256 g. Increased dosage caused a rise in the absolute concentration in tumour which, for the highest dose, reached 5.1 micrograms/g at 3 days after injection. A concomitant increase in concentration of 1H12 in blood occurred, which with the highest dose, remained above that in the tumour up to 7 days after injection. With F(ab')2 fragments (prepared from anti-CEA, 1C12) increased doses up to 380 micrograms also resulted in an increased uptake in tumour reaching almost 3 micrograms/g for a 234-micrograms dose. Circulatory clearance of F(ab')2-1C12 was essentially complete by 2 days for all doses up to 234 micrograms. Differences in clearance between 1H12 and F(ab')2-1C12 were reflected in the tumour to blood ratios. For high doses of 1H12 this ratio did not exceed unity up to 8 days. With F(ab')2, however, the tumour to blood ratio remained unaffected by dosage after 2 days. Our data suggest that F(ab')2 fragments clear sufficiently quickly to allow compensation by dosage for their premature escape from tumour. Therapeutic administration of intact antibody, however, appears to be limited by a protracted excretory process.     

Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier.

Monoclonal antibodies (MAbs) often distribute nonuniformly in tumors. In part, that observation reflects intrinsic heterogeneity within the tumor; in part, it reflects poor penetration through tumor substance. Several years ago, we proposed the "binding site barrier" hypothesis (J.N. Weinstein, R.R. Eger, D.G. Covell, C.D.V. Black, J. Mulshine, J.A. Carrasquillo, S.M. Larson, and A.M. Keenan, Ann. NY Acad. Sci., 507: 199-210, 1987; K. Fujimori, D.C. Covell, J.E. Fletcher, and J.N. Weinstein, Cancer Res., 49: 5656-5663, 1989), the idea that antibodies (and other ligands) could be prevented from penetrating tumors by the very fact of their successful binding to target antigen. Calculations suggested that this might be a significant factor in the therapy of even microscopic nodules. The higher the affinity and the higher the antigen density, the greater the barrier. Here, we provide direct experimental evidence of such a barrier to the percolation of D3 MAb through intradermally implanted line 10 carcinoma of guinea pigs. After affinity purification using glutaraldehyde-fixed line 10 cells, the D3 had an average immunoreactivity of 88%, a binding constant of 1.6 +/- 0.3 (SEM) x 10(10) M-1, and saturation binding of 355,000 +/- 15,000 molecules/cell. Using a combination of double-label autoradiography and double-chromagen immunohistochemistry, we determined simultaneously the distribution of (a) i.v. injected D3 MAb; (b) coinjected isotype-matched control IgG (BL3); (c) D3 antigen; (d) blood vessels. The previously developed mathematical models aided in the design of these experiments. Double immunochemical staining of the tumors showed antigen-rich patches 100-800 microns across, surrounded by blood vessels. At a low MAb dose (30 micrograms), binding to antigen severely hindered penetration into antigenic patches as small as 200 microns, even at 72 h. Explanation of this finding by a physical barrier was ruled out by the observation that BL3 distributed uniformly in the same patches. At a higher dose (1000 micrograms), the binding site barrier could be partially overcome. The same general principles of micropharmacology may apply to biological ligands other than antibodies, including those secreted by genetically modified cells.     

Pharmacokinetics of radiolabeled monoclonal antibody B6.2 in patients with metastatic breast cancer

Thirteen patients with metastatic breast carcinoma were given injections of 50-1593 micrograms of 131I-monoclonal antibody (MAb) B6.2 immunoglobulin G and F(ab')2 for pharmacokinetic evaluation and radioimmunoimaging. Blood clearance of the 131I-MAb-B6.2 was biphasic. The mean half-times (t 1/2 alpha, t 1/2 beta) for the immunoglobulin G were 3.5 +/- 1.7 and 20.9 +/- 11.0 h, respectively. The t 1/2 alpha for the F(ab')2 was 1.7 +/- 1.3 h, and the t 1/2 beta was 31.0 +/- 5.7 h. The percentage of protein bound 131I for the immunoglobulin G and for the F(ab')2 at 72 h was 73.7 +/- 11.4% and 58.2 +/- 14.5%, respectively. In vitro reactivity of MAb B6.2 with granulocytes isolated from normal subjects and patients was demonstrated by cytofluorometric and radioimmunoassays. MAb B6.2 was shown to bind with normal cross-reacting antigen, a cell surface antigen known to be expressed on normal human granulocytes. Reactivity with normal cross-reacting antigen on granulocytes is consistent with the skeletal images obtained during immunoscintigraphy of all 13 patients. A specific tumor image was observed in one patient. No toxicity was encountered. In spite of extensive preclinical data suggesting that 131I-MAb B6.2 would be a useful agent for radioimmunoimaging, the clinical utility of this reagent is probably limited because of the reactivity with granulocytes.     

Localization of radiolabeled antimyeloid antibodies in a human acute leukemia xenograft tumor model.

Acute myeloid leukemia is an attractive disease to treat with radiolabeled antibodies because it is radiosensitive and antibody has ready access to the marrow cavity. In order to evaluate potentially useful radiolabeled antibodies against human acute myeloid leukemia, we have developed a nude mouse xenograft model using the human acute leukemia cell line, HEL. Mice with s.c. xenografts of HEL cells received infusions of radioiodinated anti-CD33 antibody. Examination of the biodistribution of the antibody showed that uptake in the s.c. tumor was maximal [16.9% injected dose (ID)/g at 1 h after infusion] following infusion of 1-10 micrograms of antibody and decreased following infusion of 100 micrograms (6.5% ID/g at 1 h) presumably as a result of saturation of antigen sites. The radiolabel was poorly retained in tumor (4.5-8.2% ID/g at 24 h after infusion). These results were consistent with in vitro studies demonstrating rapid internalization and catabolism of the anti-CD33 antibody. Uptake in tumor could be improved by using either a radiolabel that is retained intracellularly, 111In-DTPA (18.5% ID/g at 24 h), or by targeting a surface antigen that does not internalize upon antibody binding, CD45 (20.5% ID/g at 24 h). These results indicate that this model system will be useful in evaluating the interaction of radiolabeled antibodies with human acute myeloid leukemia cells in an in vivo setting.     

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 and F(ab')2 fragment delivery to tumor in patients with glioma: comparison of intracarotid and intravenous administration

Non-i.v. delivery of radiolabeled monoclonal antibodies (MAbs) has been shown to increase tumor uptake and decrease dose to normal tissues. In this study, we have examined the potential advantage of intracarotid (i.c.) versus i.v. administration for the delivery of an intact MAb and a F(ab')2 fragment to tumor in patients with gliomas. Three patients received 10-50 mg of 81C6 IgG2b, a MAb reactive with the glioma-associated extracellular matrix antigen tenascin, and three received 5-20 mg of the F(ab')2 fragment of Mel-14, which is reactive with gliomas and melanomas. Paired-injection protocols, in which one-half of the MAb was labeled with 131I and administered by i.c. injection, and one-half was labeled with 125I and simultaneously administered by i.v. injection, were used. For both 81C6 IgG2b and Mel-14 (Fab')2, no differences in blood clearance half-times or urinary excretion rates of radioiodine were observed between i.c.- and i.v.-administered activity. Analysis of biopsy samples revealed i.c.:i.v. uptake ratios of 1.02 +/- 0.04, 0.95 +/- 0.03, and 1.03 +/- 0.05 for the accumulation of 81C6 IgG2b in temporalis muscle, normal brain, and glioma, respectively. Similarly, the i.c.:i.v. uptake ratios for Mel-14 F(ab')2 in these tissues were 0.98 +/- 0.04 (SD), 1.00 +/- 0.05, and 1.04 +/- 0.05. When the differences in percentage of injected dose/g uptake after i.c. and i.v. administration were compared, no statistically significant advantage for i.c. delivery was seen (P = 0.22-0.61). These data indicate that i.c. administration of MAb 81C6 IgG2b and Mel-14 F(ab')2 fragments offers no delivery advantage to offset the small but finite risk involved in cannulation and injection of the internal carotid artery.     

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.     

Selective radiation of hematolymphoid tissue delivered by anti-CD45 antibody.

The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.     

Comparison of therapeutic efficacy and host toxicity of two different 131I-labelled antibodies and their fragments in the GW-39 colonic cancer xenograft model

The in vivo uptake, therapeutic potential, and host toxicity were evaluated for both the intact IgG and F(ab')2 fragment of 2 murine monoclonal antibodies (MAbs) directed against either carcino-embryonic antigen (CEA), called NP-4, or colon-specific antigen-p (CSAp), called Mu-9, in the GW-39 human colorectal carcinoma grown in the hamster cheek pouch. Mu-9 IgG and F(ab')2 were retained longer by the tumor than was the NP-4 IgG or F(ab')2, respectively. Localization of the two antibodies by micro-autoradiography revealed two distinct patterns. Mu-9 was seen densely around whole acini of tumor cells, whereas NP-4 was found around each individual cell, albeit less densely. The anti-tumor effects of 131I-labelled Mu-9 and NP-4 IgG were equal, but the therapeutic effectiveness of Mu-9 F(ab')2 was significantly higher than NP-4 F(ab')2, as measured by change in tumor size. A dose-dependent increase in host toxicity, as measured by change in body weight and change in peripheral white blood cells (p-WBCs), was observed with 0.5 to 3.0 mCi doses of 131I-IgG regardless of the MAb used. A 10-22% loss in body weight lasting 3-7 weeks and a 50-80% loss in pWBCs lasting 6-8 weeks were observed in these animals. In contrast, 3 x 2 mCi doses of 131I-NP-4 or Mu-9 F(ab')2 given at 3-day intervals, a schedule which was equally therapeutic to a single 2-mCi dose of 131I-IgG, resulted in only a 9% loss in body weight and a 50% loss in pWBCs that lasted only 1 week. This was followed by a complete recovery over the next 2-3 weeks. These data suggest that multiple doses of F(ab')2 can be as tumoricidal as a single dose of an intact MAb IgG, but significantly less toxic to the host.     

Therapy of colorectal carcinoma with monoclonal antibodies (MAb17-1A) alone and in combination with granulocyte monocyte-colony stimulating factor (GM-CSF).

A mouse monoclonal antibody (MAb17-1A) (IgG2A) against colorectal carcinoma cells was used to treat patients with metastatic disease. Major direct effector functions of MAb seem to be ADCC (antibody dependent cellular cytotoxicity), CDC (complement dependent cytolysis) and apoptosis ('programmed cell death'). Thus, a high tumor cell saturation of the MAb should be achieved. Increasing doses of MAb to the patients increased the total area under the concentration curve and thus the exposure of tumor cells to MAb. However, the response rate (with complete + partial + minor response + stable disease defined as response) was not augmented. In total, 10/52 (19%) patients responded and in fact lower doses (less than 2 g) might induce a higher response frequency (9/52) than higher doses (greater than 2 g) (1/52). During treatment, the numbers of cytotoxic cells (lymphocytes and monocytes) increases in the tumor lesion and complement components were deposited. As ADCC may be important, effector mechanism attempts were made to augment the cytolytic capability of the effector cells by simultaneously giving the patients GM-CSF. The combination of MAb17-1A + GM-CSF augmented the ADCC activity of blood mononuclear cells and a heavy infiltration of monocytes could be noted in the tumor. Out of 15 available patients 6 (40%) showed a response.     

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.     

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

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

Quantitative and qualitative aspects of radiolocalization in colon cancer patients of intravenously administered MAb B72.3

Monoclonal antibody (MAb) B72.3 has been previously shown, by in vitro assays, to have a high degree of specificity for carcinomas of the colon, ovary and breast versus normal adult tissues. B72.3 IgG was labelled with 131I and injected i.v. into 20 patients with known or suspected colorectal cancer. All patients subsequently underwent surgical exploration, with tumor and selected normal tissues removed for staging purposes. The selective localization of 131I-MAb B72.3 IgG was demonstrated in biodistribution studies in which the % ID/g of each tumor was compared with that of the normal tissues, thus providing a relative RI for each lesion. Of the tumor lesions, 70% (99/142) had an RI of at least 3 (i.e., 3 times greater uptake per gram than normal tissues), and 31% of the tumor lesions had RIs of over 10. Only 12 of 210 (6%) histologically normal tissues had RIs of greater than 3; either these tissues were adjacent to or draining tumor masses or, as in the case of 2 patients, the high RI values were apparently due to deposition of immune complexes in the splenic tissues. Several parameters were studied to determine factors that might influence MAb localization. Whereas tumors of all histologic types localized the MAb, 31% of the well-differentiated mucinous carcinomas displayed tumor-to-normal ratios greater than 10, while less than 5% of the lesions of other tumor types demonstrated similar localization. The expression of the antigen (TAG-72) detected by MAb B72.3 in these tumors, as studied by immunohistochemical techniques using tissue sections, did not always correlate with the outcome of the MAb distribution. No differences in MAb uptake were observed among the carcinoma lesions from numerous anatomic locations, demonstrating the ability of i.v. administered B72.3 to reach all the tumor sites. Furthermore, autoradiographic studies of tumors showed good penetration of the MAb into the medial areas of the tumors, regardless of their size.     

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model

Monoclonal antibodies (Mab) directed against distinct epitopes of the human 240 kD melanoma-associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I-labeled MAb Me1-14. This ratio was further increased to 43 and 23 by the use of F(ab')2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab')2 fragments from MAb Me1-14 and anti-CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab')2 fragment from MAb Me1-14 is suitable for melanoma detection by immunoscintigraphy in patients.     

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

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