The effects of circulating antigen on the pharmacokinetics and radioimmunoscintigraphic properties of 99m Tc labelled monoclonal antibodies in cancer patients.

UNLABELLED: PURPOSE. This article reports the pharmacokinetics, radiation dosimetry and radioimmunoscintigraphy (RIS) of two (99m)Tc-labelled monoclonal antibodies (MAb) used to detect cancer. METHODS: The effects of circulating antigen in female cancer patients are explored and their effects on the ability of these MAbs to effectively perform as RIS agents noted. To illustrate the effects of circulating antigen, data using MAb B43.13 (OVAREX, AltaRex Corp., Waltham, MA, USA) from a Pilot study in ovarian cancer patients are presented. The results from a Phase II study of MAb 170H.82 (Tru-Scint AD, BIOMIRA INC., Edmonton, Alberta, Canada) in patients with primary and locally recurrent breast cancer were used to portray the biodistribution patterns when no circulating antigen is present. Data from planar gamma camera images were obtained for both groups and used for pharmacokinetic and radiation dosimetry analyses. RESULTS: A pharmacokinetic analysis indicated a shorter residence time and higher clearance of (99m)Tc-MAb-B43.13 that was ascribed in part to the circulating CA 125 antigen in this group of ovarian cancer patients. CONCLUSION: These clearance patterns resulted in acceptable, though higher radiation doses to the spleen and urinary bladder wall for these patients when compared to the MAb-170H.82 group. Both MAbs were found to produce acceptable radioimmunoscintigraphic images

Pharmacokinetics and radiation dosimetry of 99Tcm-labelled monoclonal antibody B43.13 in ovarian cancer patients.

OVAREX MAb B43.13 is a new radiopharmaceutical based on a monoclonal antibody (MAb-B43.13) known to recognize CA 125, a tumour antigen associated with epithelial ovarian cancer. This MAb is capable of facile radiolabelling with 99Tcm and has been shown previously to localize in the tumours of ovarian cancer patients. The present study was initiated to measure the pharmacokinetics of this MAb in the serum of 10 patients with primary or metastatic ovarian cancer. A two-compartment model was found to be best at representing the biodistribution of the 99Tcm-labelled MAb, yielding a 2.6 h distribution phase half-life and a 31.3 h elimination phase half-life. The serum and renal clearances for 99Tcm-MAb-B43.13 were 121 and 53 ml h-1 respectively. These parameters were compared with a similar model developed from the serum values of the MAb itself (determined using an ELISA detection method). Based on the serum pharmacokinetics of 99Tcm-MAb-B43.13 and whole-body planar gamma camera images, an estimate of the radiation dose from 99Tcm was calculated using standard MIRD schema. The organs demonstrating significant 99Tcm uptake included the liver, kidneys, heart and spleen. The whole-body dose was similar to other 99Tcm-labelled MAbs.

Radiolabeling of monoclonal antibody B43.13 with rhenium-188 for immunoradiotherapy.

In this study we report a novel method for direct radiolabeling of monoclonal antibody B43.13 (MAb-B43.13) with 188Re and have evaluated the product's radiochemical, biochemical, immunochemical and selected biological properties. 188Re-MAb-B43.13 was readily prepared by the addition of generator produced perrhenate to a preformulated antibody vial after an optimal amount of supplemental stannous ion, in the form of stannous tartrate, was added. The final radiolabeled product retained its biochemical purity (as determined by size-exclusion HPLC and R/NR-SDS-PAGE), its immunoreactivity (as determined by immunoassay) and presented with a typical stability (in the presence of serum and cysteine) and biodistribution (in tumored mice) profile. The evaluation of the product for immunoradiotherapy of ovarian cancer in a clinical setting requires further studies.

Role of transchelation in the uptake of 99mTc-MAb in liver and kidney.

High radioactivity in liver and kidney after administration of 99mTc-labeled antibodies is a major detriment to the use of radiolabeled antibodies for diagnosis and therapy. In the present study, the uptake mechanism of radioactivity by liver and kidney involving 99mTc moiety was investigated. The data of in vitro and in vivo thiol transchelation studies, biodistribution alteration of 99mTc-MAb after specific modulation of endogenous thiol containing compounds, and the finding of 99mTc-labeled cysteine and GSH in bile, urine and kidney after administration of 99mTc-MAb demonstrated that transchelation by thiols (cysteine and GSH) played an important role in the localization of radiotracer from 99mTc MAb in normal tissues such as liver and kidney.

Studies on metabolism of directly labeled 99mTc-antibody in mice.

The elucidation on the metabolic products of the 99mTc-antibody conjugates may provide insights and approaches that would reduce the undesirable deposition of radioactive species in normal tissues. In this investigation, the radiolabeled species in blood, urine, bile and extracts of liver and kidney obtained at different times after the injection of a model antibody, 99mTc-MAb170, into mice were analyzed with various chromatographic methods. Ninety-nine to 100% of the radioactivity in serum was associated with intact MAb170. The radioactivity in liver homogenate extract was strictly protein-bound to either intact MAb or low molecular weight species (LMW). In kidney extracts, the majority of the radioactivity was protein bound 99mTc, with less than 8% of the activity being non-protein bound 99mTc. Multiple 99mTc-containing protein and non-protein species were found in urine and bile. Evidence supporting the presence of 99mTc-cysteine and 99mTc-glutathione in bile, kidney and urine was also obtained. No evidence for the in vivo formation of 99mTc-pertechnetate in mouse blood, liver, kidney, bile and urine was observed.

Preparation and protein conjugation of a divinyl sulphone derivatized bifunctional chelating agent.

A new bifunctional chelating agent with a novel linking arm, 2-[p-¿N-benzyl-N-(2-vinylsulfoethyl)¿- (aminobenzyl)¿-1,3-propane-diamine-N,N,N',N'-tetraacetic acid (VS-PDTA) was synthesized and was conjugated to protein for the purpose of attaching radiometals to monoclonal antibodies (MAbs). The effect of various parameters such as ligand concentration, protein concentration, pH, temperature and reaction period on the conjugation have been examined using chromatographic (SE and TLC) analysis after labeling with 111In. The parameters and chemical variables studied have significant effects on the efficiency and rate of protein conjugation.

Studies on the uptake mechanisms of 99mTc-labeled antibodies by liver tissue.

In this study, the roles of various protein alterations on the uptake of 99mTc labeled antibodies by isolated hepatocytes and by selected tissues in vivo were explored. In vitro binding studies of radiolabeled antibodies with hepatocytes demonstrated that the uptake of the radiolabel was a function of incubation duration and dose dependent. The uptake of 99mTc-antibodies could be inhibited by excess unlabeled F(ab')2 and Fc fragments as well as intact antibody. Liver uptake could not be reduced by a large insert pretreatment dose of unlabeled or aggregated antibody. These observations indicated that both Fab and Fc portions on the antibody molecule as well as the intact antibody may play important roles in the uptake of the radiolabel by liver tissue.

Synthesis and evaluation of a new bifunctional chelating agent for the preparation of radioimmunoconjugates.

The conjugation of radiometals to monoclonal antibodies results in agents for radioimmunoimaging and other medical applications. Due to remarkable ability to form stable metal complexes with a great number of metal ions in different oxidation states, polyaminocarboxylate chelates are useful tools for this purpose. Bifunctional chelators that can hold radiometals with high stability under physiological conditions are essential to avoid radiation damage to non-target organs. We have synthesized a new bifunctional chelate 2-(p-aminobenzyl)-1,3-propylenediamine-N,N,N',N'-tetraacetic acid by a simple method and studied the rate of loss of radioactivity from the radiolabeled (111In, 90Y) chelates to serum proteins in human serum at 37 degrees C. The relative stability constant of this new bifunctional chelate was found to be very similar to the underivated form. This chelate was conjugated to murine monoclonal antibody (B43) and immunoreactivity of the conjugated was determined by competitive binding analysis, which showed no significant change in its immunological activity. Biodistribution of the 111In radioconjugate was examined in conventional Balb/c and tumor-bearing (-OVCAR-3) athymic Balb/c mice.

Direct labeling of monoclonal antibodies with technetium-99m by photoactivation.

UNLABELLED: Direct radiolabeling methods currently rely on the addition of exogenous chemical reagents to create the necessary binding sites for 99mTc binding to monoclonal antibodies (MAbs). This work describes the use of ultraviolet (UV) light to facilitate photoactivation of MAbs for 99mTc radiolabeling. METHODS: The parameters of exposure wavelength and solution composition were investigated to provide a basis for further development. Based on these results, various murine MAbs and a chimeric MAb were photoactivated using a 300-nm (nominal) wavelength, eight-lamp (3.9 W each) photochemical reactor providing exposure for defined time periods. The MAb preparations were stored frozen and subsequently labeled by the addition of pertechnetate. For MAb-170, the photoactivated preparation was compared to a stannous ion reduced preparation by radiochemical (radiolabeling yield, serum stability cysteine challenge), biochemical (SDS-PAGE, IEF, SE-HPLC) and immunochemical (immunoreactivity) assays and biodistribution studies in mice. RESULTS: Photoactivation produced high radiolabeling yields for all the MAbs studied and MAb-170 produced comparable in vitro quality control profiles and in vivo biodistribution data. CONCLUSION: The use of this relatively simple, short and easily controlled photoactivation process for MAbs facilitates facile radiolabeling with 99mTc and provides an alternative to the direct chemical radiolabeling procedures.

Antiidiotype induction therapy: evidence for the induction of immune response through the idiotype network in patients with ovarian cancer after administration of anti-CA125 murine monoclonal antibody B43.13.

The immune status of ovarian cancer patients receiving anti-CA125 murine monoclonal antibody B43.13 was evaluated by measuring antiidiotypic antibodies (Ab2), antiantiidiotypic antibodies (Ab3), antiisotypic human antimouse antibodies (HAMA), interferon-gamma, and CA125 levels in the serum. A specific assay was developed for the determination of Ab2 antibodies using chimeric MAb B43.13. Of the 50 patients studied, 26 had elevated levels of Ab2. Eleven of these 26 patients also had high titer of antiantiidiotypic (Ab3) antibodies. Eight of the 22 patients analyzed had increased interferon-gamma levels. A tentative correlation was found between survival of these patients' antiidiotype induction.

An improved method for analysis of technetium radiolabeled antibody by size exclusion HPLC.

A quantitative radio HPLC method for the analysis of radiolabeled 99mTc compounds, in particular labeled antibodies or proteins, is described. The method is based on the quantitative re-oxidation of the reduced column absorbed 99mTc species to pertechnetate using hydrogen peroxide. The pertechnetate is quantitatively eluted from the column. The method can be used as a stand alone technique for determining the true radiolabeling yield of labeled proteins/antibodies. The method has been automated for routine use and the typical analysis time is 30 min.

Activating anti-idiotypic human anti-mouse antibodies for immunotherapy of ovarian carcinoma.

Human anti-mouse antibodies (HAMA) are observed frequently after immunoscintigraphy with monoclonal antibodies (MoAb) directed against CA-125. As the authors have shown previously, HAMA can cause false-positive CA-125 values in routine CA-125 immunoradiometric assay (IRMA) tumor-marker assays (in one case, up to 900 days after immunoscintigraphy). In 32 patients, the authors found a HAMA frequency of 34% (11/32: 3/7 after the first administration, 6/13 after the second, and 2/2 after the third). Ten patients developed extremely high CA-125 levels after undergoing the CIS IRMA assay (up to 80,000 U/ml) in parallel to a significant HAMA increase. The use of different assays, or HAMA removal before in vitro testing, can solve this problem. After a new CA-125 assay containing antibodies that recognize different epitopes on the CA-125 antigen (Biomira Tru-Quant OV) was applied, only mildly increased assay results or normal levels were measured. Most of HAMA-positive patients demonstrated a predominantly anti-idiotypic response, determined with two different HAMA assays. Seven patients with anti-idiotypic HAMA responses after OC-125 immunoscintigraphy remained free of tumor or had stable disease (2-42 or more months), contrary to their poor prognoses that had been made based on the underlying stages of their tumors. All of these patients are currently doing well (Karnofsky Index > 70%) and show no significant tumor progression. In light of their extremely poor prognoses (5-year survival rates of 3-5% in recurrent International Federation of Gynecology and Obstetrics III/IV stages), without further chemotherapy, these courses are extremely unusual. Preliminary in vitro experiments lead to the postulation that anti-idiotypic HAMA may trigger an antitumor effect either by suppressing the growth of CA-125-expressing cancer cells directly, or by activating the patient's immune response via induction of Ab3. Similar results are observed after immunoscintigraphy with a technetium-99m-labeled anti-CA-125 monoclonal antibody (B43.13), which the authors now also use for immunotherapy of ovarian cancer patients by repeated injections, hoping that induction of anti-idiotypic HAMA will be beneficial for prolonged survival of patients with ovarian carcinoma.

Preparation and biological evaluation of radiolabelled antibodies with selected carbohydrate modifications.

Two carbohydrates, N-acetylgalactosamine (GalNAc) and galactose-beta-1,3-GalNAc have been attached to human IgG (hIgG) by a novel linking reagent, hexafluoroglutaric acid dimethyl ester. Fluorine-19 NMR signals were used for the determination of the conjugation ratio. A third carbohydrate, sialic acid, was conjugated via reductive amination and the conjugation ratio determined by a resorcinol assay. The biological behaviour of these radioiodinated antibodies with carbohydrate modifications in normal mice indicates an enhanced liver uptake at 15 min post-injection with an associated change in circulating blood levels occurs for the galactose-based hIgG preparations. However, no significant differences in the biodistribution were observed for the sialic acid conjugate. These studies confirm the potential of carbohydrate-antibody conjugation for modifying the behaviour of antibodies in immunoscintigraphy and radioimmunotherapy.

Stability of ganciclovir sodium in 5% dextrose injection and in 0.9% sodium chloride injection over 35 days.

The stability of ganciclovir 1 and 5 mg/mL in 5% dextrose injection and in 0.9% sodium chloride injection was studied at 25 degrees C and 5 degrees C over 35 days. Ganciclovir (as the sodium salt) was added to 120 polyvinyl chloride bags containing either 5% dextrose injection or 0.9% sodium chloride injection to attain ganciclovir concentrations of 1 and 5 mg/mL. Thirty bags were prepared for each combination of drug concentration and i.v. solution. Half of the bags in each group were stored at 25 degrees C; the other half were stored at 5 degrees C. Samples withdrawn from all 120 bags immediately after preparation were frozen for later determination of initial concentration. At 7, 14, 21, 28, and 35 days after preparation, approximately 5-mL samples representing each test condition were withdrawn for analysis. The samples were visually examined, tested for pH, and assayed by high-performance liquid chromatography. There was no significant loss of ganciclovir under any of the study conditions over 35 days. All solutions were clear throughout the study period. The pH decreased slightly in both diluents at both ganciclovir concentrations but did not deviate from the manufacturer's range (9-11). Admixtures containing ganciclovir 1 and 5 mg/mL (as the sodium salt) in 5% dextrose injection and 0.9% sodium chloride injection were stable in polyvinyl chloride bags stored at 25 degrees C and 5 degrees C for 35 days.

Comparative radiolabeling and distribution of a tumour-directed monoclonal antibody.

The monoclonal antibody (MAb) 155H.7, raised against a synthetic beta-anomer of the Thomsen-Friedenreich antigen (S-TAG), was radioiodinated using iodine monochloride, chloramine-T and Iodogen and radiolabeled with 111In using the bromoacetamido-derivative of benzyl-EDTA. The in vitro immunoreactivity of the MAb was assessed using an ELISA with the S-TAG and the in vivo distribution of the radioiodinated and radiochelated MAb was determined in the murine mammary carcinoma TA3/Ha tumour model. Both chloramine-T and iodine monochloride radioiodination greatly reduced the immunoreactivity of the MAb compared to radioiodination using Iodogen. Bifunctional chelate labeling was comparable to Iodogen in reducing the immunoreactivity of the MAb and subsequent chelation of 111In did not further compromise the immunoreactivity of the MAb. The in vivo distribution data showed significantly different distributions of the radiolabels after injection of the radioiodinated and radiochelated MAb. The 131I-MAb showed some tumour association as compared to the distribution of an 125I-non-specific protein and the data also indicates that there is preferential dehalogenation of the radioiodinated MAb. 111In from the radiochelated MAb showed significantly higher uptake in the tumour than 131I from the 131I-MAb. It is suggested that the differing fates of the two radiolabels within the tumour cell is responsible for the difference in retention observed and not necessarily due to the lack of MAb uptake by the tumour. Overall, the radiochelate label for MAb 155H.7 appears to be superior to radioiodine for in vivo use.

Biological behavior of 67Ga-citrate in New Zealand White rabbits.

The pharmacokinetics, protein binding, excretion and tissue distribution of 67Ga after the administration of 67Ga-citrate to New Zealand White rabbits is described. Data for 67Ga blood levels were best described by an equation with three exponential components exhibiting half lives of 0.25 h, 7.4 h and 19.5 h, with almost all of the activity in a protein bound form. Weekly urinary excretion (approximately 27%), possibly in a metabolized form, and fecal elimination (approximately 20%) were greater than the reported values in man, but there was a similar organ distribution pattern in these animals as in man. The overall biological handling was judged to be similar in both species making the rabbit a suitable model for further 67Ga-citrate studies in vivo.

Plasma protein fraction and albumin use: the effect of medical staff education.

A 6-week audit evaluating the use of plasma protein fraction and albumin was conducted and study data demonstrated that these colloids were being inappropriately prescribed. The P & T Committee decided to use educational rather than restrictive methods to improve colloid prescribing. Follow-up audits were performed, one immediately following staff education and another 1 year after the first audit. Although the educational actions employed in this drug utilization review appeared to have an immediate impact, the effect was not sustained long-term, indicating the need for periodic medical staff education. Methods of effective educational intervention as reported in the literature are discussed.

Effect of cis-platinum on the protein binding and pharmacokinetics of 67Ga-citrate.

Cis-platinum, a widely used chemotherapeutic drug, and K2PtCl4, a related complex, were found to inhibit the in vitro protein binding of 67Ga to human transferrin in isolated solution but not to whole serum. Cis-platinum treatment of rabbits resulted in enhanced protein binding and delayed blood clearance of subsequently injected 67Ga-citrate. These effects are related to the toxicity of the drug in vivo.

Immediate and delayed effects of cis-platinum treatment on the short term biodistribution of 67Ga-citrate in mice.

The co-administration of a single medium toxic dose (5 mg/kg) of the chemotherapeutic drug cis-platinum (cis-Pt) with 67Ga-citrate was found to have minimal effects on the 3 h tissue distribution of the radiopharmaceutical in mice. When the same drug dose was given at two day intervals preceding the co-administered dose, virtually all tissues accumulated 67Ga to a greater extent. The tissue distribution of radiolabeled cis-Pt supported the development of drug-induced renal dysfunction as the primary cause.

Effect of cis-platinum treatment on 67Ga-citrate localization and elimination in rabbits.

The treatment of rabbits with a typical dosage schedule of the chemotherapeutic drug cis-platinum resulted in an increased whole body retention of 67Ga-citrate due to decreased urinary and fecal excretion. The biodistribution of the tracer was also affected with most tissues showing greater accumulation. These changes are primarily related to drug-induced renal dysfunction, but other factors may also be involved.