Phase I study of the pharmacokinetics of a radioimmunoconjugate, 90Y-T101, in patients with CD5-expressing leukemia and lymphoma.

Ten patients with advanced or refractory CD5-expressing hematologic neoplasms [two with chronic lymphocytic leukemia and eight with cutaneous T-cell lymphoma (CTCL)] were treated in a Phase I study with the radioimmunoconjugate 90Y-T101, which targets CD5+ lymphocytes. Prior imaging studies using 111In-T101 demonstrated uptake in involved lymph nodes and skin in patients with CTCL, and Phase I studies with unmodified T101 demonstrated transient responses. In this study, patients were treated with 5 or 10 mCi of 90Y chelated to T101 via isothiocyanatobenzyl diethylenetriamine pentaacetic acid, along with tracer doses of 111In-T101 for imaging. The biodistribution of the radioimmunoconjugate was determined by measuring 90Y and 111In blood clearance, urine excretion, and accumulation in bone marrow and in involved skin lesions. The intravascular pharmacokinetics of 90Y were predicted by 111In-labeled T101. The greatest differences in biodistribution between 111In and 90Y were in the higher bone accumulation of 90Y and its lower urinary excretion. Imaging studies demonstrated targeting of skin lesions and involved lymph nodes in CTCL patients. The predominant toxicity was bone marrow suppression. Rapid antigenic modulation of CD5 on circulating T and B cells was observed. Recovery of T-cell populations occurred within 2-3 weeks; however, suppression of B-cell populations persisted after 5+ weeks. All CTCL patients developed human antimouse antibody after one cycle and thus were not retreated; one patient with chronic lymphocytic leukemia received a second cycle of therapy. Partial responses occurred in five patients, two with chronic lymphocytic leukemia and three with CTCL. The median response duration was 23 weeks. One CTCL patient who subsequently received electron beam irradiation to a residual lesion is disease-free after 6 years.

Bifunctional antibody: a binary radiopharmaceutical delivery system for imaging colorectal carcinoma.

In clinical studies we have evaluated a unique monoclonal antibody-based drug delivery system, a bifunctional antibody designed to deliver imaging or therapeutic agents, such as radioisotopes, drugs, or biologics, to tumor cells, while minimizing the dose to normal tissue. The bifunctional antibody, with one specificity to a tumor-associated antigen (carcinoembryonic antigen) and another specificity to a hapten, is injected and allowed to localize at a tumor site for 4 days. A hapten, tagged with a radioisotope, is subsequently injected for delivery to and capture by the prelocalized antibody at the tumor site. In studies reported here, the sulfhydryl groups of Fab' fragments of ZCE-025 and CHA-255 were linked with bis-maleimidomethyl ether to form an F(ab')2 bifunctional antibody coupled by a stable thioether linkage. EOTUBE, a hydroxyethylthiourido derivative of benzyl EDTA, was used as the hapten carrier of 111In. Fourteen patients 62-82 years old with recurrent or metastatic adenocarcinoma of the colon were studied. Twenty of 21 known lesions were imaged, and eight of nine new lesions were confirmed. With this fundamentally new approach to drug delivery, clearance from normal tissue is rapid, and high tumor:normal tissue ratios are expeditiously achieved.

Kinetics of the dissociation of indium-(p-substituted-benzyl)ethylenediaminetetraacetic acid hapten analogues from the monoclonal anti-hapten antibody CHA255.

Half-lives were measured for the dissociation of a series of 20 indium-benzyl-EDTA derivatives from a monoclonal antibody that binds to them. Most haptens gave expected monoexponential dissociation curves with half-lives ranging from approximately 8 to approximately 100 min at 22 +/- 1 degree C. Precise (+/- approximately 2.5%) determinations were made using centrifugal ultrafiltration to separate free from bound hapten. A strong pH dependence of the dissociation half-life was found for the two haptens studied. Activation enthalpies were identical (23 +/- 1 kcal/mol) for the dissociation of four haptens, suggesting that, in contrast to individual rate constants, this parameter is insensitive to hapten modification. The dissociation half-lives provided evidence for the location of a positive charge in the binding site, but gave no clear indication of the role of hydrophobic interactions or of steric requirements in hapten binding. While variations in ionic strength had no effect on the dissociation rate, lowering surface tension with dioxane increased the rate somewhat. Three hapten-antibody complexes showed biexponential dissociation rates. It is postulated that this results from distinct conformations of the complex dissociating at different rates. The dissociation rate constant was found to be an extremely sensitive indicator of the hapten-antibody interaction that can be measured very precisely.

Metastatic colorectal cancer: radioimmunoscintigraphy with a stabilized In-111-labeled F(ab')2 fragment of an anti-CEA monoclonal antibody.

Metastatic colorectal cancer was detected with stabilized F(ab')2 fragments of ZCE-025, an anti-carcino-embryonic antigen (CEA) monoclonal antibody (MoAb). The fragments were prepared by cross-linking Fab' with a bifunctional cross-linking agent, bis-(maleimido)methyl ether. The authors labeled 2 mg of ZCE-025 with 5 mCi (185 MBq) of indium-111 and injected the material intravenously, either alone or with unlabeled F(ab')2, into 16 patients. Lesion detection, pharmacokinetics, and relative body distribution were evaluated and compared with those of the intact immunoglobulin (IgG1) antibody. Stabilized F(ab')2 fragments were more useful than the intact antibody in detection of lesions: Overall sensitivity of F(ab')2 fragments for all the patients was 79.4%, whereas overall sensitivity of the intact IgG1 antibody was 32%. This anti-CEA-stabilized F(ab')2 fragment may be a powerful diagnostic tool that can achieve higher sensitivity at smaller protein doses than the intact IgG1 antibody.

Differential cellular catabolism of 111In, 90Y and 125I radiolabeled T101 anti-CD5 monoclonal antibody.

We evaluated the differences in catabolism of 125I, 111In and 90Y-T101 monoclonal antibody (anti-CD5) by peripheral blood mononuclear cells (PBMNC), HUT 102, CCRF-CEM and MOLT-4 cells. All cells showed higher retention of 111In than of 125I. PBMNC showed similar retention of 90Y and 111In. The release of 125I was reduced by using metabolic inhibitors, F(ab')2 of T101, pure lymphocytes or incubating at 4 degrees C. Our findings suggest differences in the intracellular catabolism of the radionuclides.

Radioimmunodetection of cancer with the use of indium-111-labeled monoclonal antibodies.

We have infused 13 111In-labeled murine IgG monoclonal antibodies (MAb) into 73 patients who had been diagnosed as having 7 types of cancers, and 3 111In-labeled human MAb into 8 patients with breast cancer. To each patient, 1.5-5 mCi attached to a maximum of 1 mg MAb had been given in a total MAb dose of 0.5-500 mg. The most encouraging overall results have been obtained with anti-human T-cell MAb T101 (33 of 33 tumor sites imaged in 5 patients), antimelanoma MAb P96.5 (47 of 88 sites imaged in 21 patients), anti-prostate MAb PSA399 (14 of 21 sites imaged in 4 patients), and anti-colon MAb ZCE025 (16 of 26 sites imaged in 12 patients). Poor imaging results were related to lower doses, reactivity with circulating cells, and limited antigen expression in various tumor sites. The problems involved in radioimmunodetection included low extraction of MAb from the serum by the tumor that resulted in poor tumor uptake of the radiopharmaceutical, and high background activity in the liver, heart, spleen, and gastrointestinal tract that made imaging difficult in those areas. Heterogeneous antigen production leaves some tumor deposits without targets, and the immunogenicity of the MAb limits use of these agents repetitively in humans. Nevertheless, these early results are encouraging for their potential diagnostic and therapeutic applications.

Preclinical trials with combinations and conjugates of T101 monoclonal antibody and doxorubicin.

We investigated the potential for additive therapy for malignancy using an anti-human T-cell monoclonal antibody, T101, and the chemotherapy agent doxorubicin (DOX). We compared the efficacy of T101 alone, DOX alone, T101 and DOX covalently linked to dextran to form an immunoconjugate, T101 plus DOX mixed together and injected, T101 and DOX injected separately, and nonspecific murine IgG2A plus DOX mixed together. Inhibition of [3H]thymidine was examined in vitro, and the clinical efficacy of each treatment was tested on human T-cell tumors growing in athymic mice. In vitro experiments confirmed retention of immunoreactivity and cytotoxicity by the immunoconjugate, but it was not superior to DOX alone. In efficacy experiments, all therapeutic arms were superior to placebo treatment (P less than 0.05). However, the best results in the animal tumor model were obtained with T101 mixed with DOX, perhaps because of formation of weak complexes via hydrophobic bonds. This mixture was superior to all other treatments, both by growth curve analysis (P less than 0.05) and by analysis of complete regression of tumor (P less than 0.01). T101 mixed with DOX was superior to a mixture of nonspecific mouse immunoglobulin and DOX and superior to a combination of T101 injected i.v. and DOX injected i.p. The antitumor effect of T101 mixed with DOX was blocked by premodulating the target antigen with T101. These data suggest that further exploration into monoclonal antibody-anthracycline complexes is warranted.

Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals.

We have prepared monoclonal antibodies (MoAbs) with the specific ability to bind metal chelates such as 111In benzyl EDTA. One, 10, 50 and 100 micrograms MoAb CHA255 Kb = 4 X 10E9 was complexed with 111In BLEDTA II, BLEDTA IV, and benzyl EDTA and injected i.v. in Balb/c mice with KHJJ tumor. The biological half-life by whole body counting was profoundly altered for all three compounds; from minutes to hours with 10 micrograms; to days with 100 micrograms. Tumor uptake increased 50 fold at 24 h with increasing MoAb but satisfactory tumor concentrations (3% per g) and tumor/blood ratios (1.8:1) were obtained with an amount equivalent to 7 mg for a human. Blood level and whole body activity were decreased 30-50% within 3 h or i.v. injection of a "flushing" dose of unlabeled indium benzyl EDTA, increasing tumor/blood ratios to 50:1.

Antibodies against metal chelates.

Because monoclonal antibodies can recognize and bind to specific groups of atoms such as tumour antigens, they have promise for use in vivo as carriers of radionuclides, drugs or other appended molecules for diagnosis and treatment of disease. Attachment of metal ions to antibodies by means of bifunctional chelating agents can add the diverse nuclear, physical and chemical properties of the metallic elements to these specific binding proteins (ref. 4 and refs therein). With the ultimate aim of engineering probe-binding properties into the antibodies themselves, we have now prepared monoclonal antibodies against the EDTA chelate of indium. These antibodies show a remarkable preference for indium chelates; changing to another metal such as scandium or gallium can decrease the antibody-binding constant by more than three orders of magnitude. These antibodies also introduce a new degree of control over the biological distributions of chelated radionuclides, markedly altering their uptake in tumours and normal organs.

Modification of human leukocyte interferon pharmacology with a monoclonal antibody.

The antitumor and antiviral properties of the interferons have been well established. However, the usefulness of the interferons may be limited, in part, because of rapid clearance from the plasma and degradation by plasma or tissue enzymes. A monoclonal antibody (IFG-252.2) was developed which binds to recombinant DNA-produced human alpha-interferon (rIFN-alpha A) without measurably reducing its in vitro antiviral or antiproliferative properties. Pharmacokinetic studies of rIFN-alpha A:antibody complex in the intact, anesthetized rat showed that rIFN-alpha A activity cleared from plasma 3-fold slower than found after injection of free rIFN-alpha A. This resulted in a 15-fold increase in its calculated area under concentration curve compared to that of free rIFN-alpha A. These studies suggest that interferon bound to a monoclonal antibody may provide a means to prevent the normal clearance and degradation of free interferon and may result in prolonged antitumor and antiviral plasma activity in vivo. Furthermore, it suggests that monoclonal antibodies to various biologically active agents may be used to favorably alter their pharmacokinetics while leaving their biological activity unaltered.

Radioimmunodetection of melanoma utilizing In-111 96.5 monoclonal antibody: a preliminary report.

The murine 96.5 monoclonal antimelanoma antibody (MoAb) was labeled with In-111, and 1-20 mg were administered to 21 patients who had proved or suspected melanoma metastases. One patient was studied twice. In four patients, unlabeled 96.5 MoAb was administered prior to the radiopharmaceutical. All of the patients tolerated the procedure without toxicity regardless of the mass of MoAb administered. The scans were interpreted by two observers, one with full knowledge, the other with no knowledge of the cases. Increasing the MoAb mass or preinfusing unlabeled MoAb prior to the administration of In-111 MoAb resulted in a prolongation of the serum half time, and appeared to improve tumor detection. Lesions were best seen at 72 hours after infusion or later. In all patients who had metastatic disease, at least one tumor site was apparent. Fifty-six per cent of known lesions 1.5 cm or greater in size were detected by the physician who had knowledge of the cases when data from all doses were considered. There were eight lesions detected that were not suspected in the workup of the patient. When these are included, the detection rate rises to 61%. Forty-nine per cent were detected by the other physician. Subtraction techniques were not employed. Lesions were often better seen with single photon emission computed tomography than with planar imaging techniques. The 96.5 In-111 MoAb appears to have utility for the detection of metastatic melanoma. Further clinical evaluation of 96.5 In-111 MoAb is warranted.

Pharmacokinetics of 111In-labeled anti-p97 monoclonal antibody in patients with metastatic malignant melanoma.

Twenty-eight patients with metastatic malignant melanoma received anti-p97 murine monoclonal antibody (96.5) infused over 2 h at doses between 1 and 20 mg coupled to either 2.5 or 5.0 mCi of 111In by the bifunctional chelating agent diethyltriaminepentaacetic acid. Clearance of 111In from plasma closely fit an open, one-compartment mathematical model (r2 greater than 0.90). The overall half-life of 111In plasma was approximately 31 h and did not appear to be dependent on the total dose of antibody administered. The apparent volume of distribution of the 111In label approximated the total blood volume (7.8 +/- 0.7 liters) at the 1-mg dose and decreased to 3.0 +/- 0.14 liters at the 20-mg dose, suggesting saturation of antigenic or other extravascular binding sites at higher antibody doses. The clearance of the murine monoclonal antibody itself from plasma was measured by an enzyme-linked immunosorbent assay. The pharmacokinetics for the murine antibody in plasma also fit an open, one-compartment mathematical model. All pharmacokinetic parameters for unlabeled antibody closely paralleled those found for 111In-labeled antibody pharmacokinetics. This suggests that the 111In radiolabel remains complexed to the monoclonal antibody after in vivo administration. The cumulative urinary excretion of the 111In label over 48 h was between 12 and 23% of the total administered dose and is assumed to represent 111In-labeled chelate complex unattached to antibody. Analysis of the 111In label in spleen, liver, heart, and kidney showed that the concentration of label in liver tissue was reduced with increasing antibody doses and coincided with changes in the apparent volume of distribution. These studies show that murine monoclonal antibodies are cleared slowly from the circulation in humans and that early, rapid distribution of labeled antibody to the liver can be reduced by increasing the dose of unlabeled antibody. This may be particularly important in limiting hepatic toxicity when administering antibody coupled to drugs, radionuclides, or toxins.

Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse-human tumor models.

Monoclonal antibodies (MoAbs) against carcinoembryonic antigen were successfully radiolabeled with 111In, and the radiopharmaceutical was characterized in vitro and in normal and tumor-bearing mice. The 111In-MoAb proved to be stable in vitro and in vivo under normal conditions, although instability could be induced in vitro with large quantities of iron-free transferrin. Animal distribution studies with 111In-MoAb demonstrated tumor localization superior to 67Ga and pharmacokinetics that were highly similar to those of endogenously labeled 75Se-MoAb. The 111In-MoAb followed first-order kinetics and fit a two-compartmental model when studied in nude mice bearing human colon tumors known to express carcinoembryonic antigen. Significant quantities of radiolabel appeared in tissues other than tumor, with liver and skin having the highest concentrations. Sufficient tumor/background ratios were formed for scanning purposes. The data indicate that 111In-MoAb may prove to be effective as a radiopharmaceutical for tumor imaging.

Localization of 111In- and 125I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors.

A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either 125I or 111In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

The major metabolite of fentanyl in the horse.

Fentanyl, a potent, synthetic narcotic analgesic, has reportedly been used to "dope" racehorses. Urine was collected from a horse dosed with 70 mg of [3H]fentanyl, and the primary metabolite, a water-soluble, amphoteric compound, was isolated by high-pressure liquid chromatography and identified by spectroscopic analysis. This metabolite was found to be N-[1-(2-phenethyl-4-piperidinyl)] malonanilinic acid.

Synthesis and analgesic activity of some long-acting piperidinospiro derivatives of methadone.

A congener of methadone, in which the metabolically labile C-6 dimethylamino moiety was replaced with a piperidinospiro derivative, was reduced and acetylated. This conversion produced a marked increase in the duration of analgesia, a trend similar to that found for methadone.