Macrophyte and periphyton dynamics in a UK Cretaceous Chalk stream: the river Kennet, a tributary of the Thames.

An initial study to observe the seasonal trends and to determine the factors influencing macrophyte and periphyton growth patterns was undertaken on a representative reach of the River Kennet (UK) over a 2-year period (1998-2000). Maximum average macrophyte and average periphyton dry matter biomass recorded during the growing season were 200 and 21 g m(-2), respectively. The relationships between macrophyte and periphyton percentage cover and biomass data with physico-chemical variables were investigated. Regression analysis indicated that of the parameters measured, flow, and in the case of the dominant Ranunculus spp., solar radiation, were best able to predict macrophyte biomass and cover. The periphytic biomass within the reach was low, possibly as a result of relatively high flows and low phosphorus concentrations following the introduction of effluent treatment at the sewage works immediately upstream of the reach. Periphytic biomass was poorly correlated with the physical variables measured. This indicates that biomass is regulated by complex interactions between the physical and chemical factors, such as flow, solar radiation and phosphorus concentration. These interrelationships require further investigation.

On modelling the flow controls on macrophyte and epiphyte dynamics in a lowland permeable catchment: the River Kennet, southern England.

A new in-stream model of phosphorus (P) and macrophyte dynamics, the Kennet Model, was applied to a reach of the River Kennet to investigate the impacts of changing flow conditions on macrophyte growth. The investigation was based on the assessment of two flow change scenarios, which both included the simulation of decreasing total phosphorus concentrations from a sewage treatment works due to improved effluent treatment. In the first scenario, the precipitation and potential evaporation outputs from a climate change model (HadCM2 GGx) where input into the catchment model INCA to predict the mean daily flows in the reach. In the second scenario, the mean daily flows observed in a historically dry year were repeated as input to the in-stream model to simulate an extended low flow period over 2 years. The simulation results suggest that changes in the seasonal distribution of flow were not detrimental to macrophyte growth. However, the simulation of extended periods of low flow suggests that a proliferation of epiphytic algae occurs, even when the in-stream phosphorus concentrations are reduced due to effluent treatment. This epiphytic growth was predicted to reduce the macrophyte peak biomass within the reach by approximately 80%. Thus, the model simulations suggest that flow was more important in controlling the macrophyte biomass in the River Kennet, than the in-stream phosphorus concentrations, which are elevated due to agricultural diffuse sources.

Evaluation of pulmonary alveolar epithelial integrity by the detection of restriction to diffusion of hydrophilic solutes of different molecular sizes.

The rate of transfer of a hydrophilic solute from the alveoli to pulmonary blood following inhalation as an aerosol depends on the molecular size of the solute and the permeability of the alveolar epithelium. The value of this measurement for assessing damage to the epithelium in lung disease is compromised by cigarette smoking, which accelerates clearance by unknown mechanisms. The rates of clearance of (99m)Tc-labelled diethylenetriaminepenta-acetic acid (DTPA) (molecular mass 492 Da) and (113m)In-labelled biotinylated DTPA (B-DTPA) (molecular mass 1215 Da) were monitored simultaneously by dynamic gamma-radiation camera imaging following simultaneous inhalation, and compared between eight normal non-smoking subjects and nine habitual cigarette smokers. The clearance rates of DTPA were 0.95 (S.D. 0.39)%/min in non-smokers and 4.13 (1.06) %/min in smokers. These were about twice the clearance rates of B-DTPA, which in the corresponding groups were 0.41 (0.26) and 2.12 (0.72)%/min respectively. The ratio of the B-DTPA/DTPA clearance rates was, in all subjects, less than the ratio (0.74) of the cube roots of the molecular masses of the solutes, assumed to correspond to the ratio of their free diffusion coefficients in water, and was not significantly different between smokers and non-smokers. As alveolar permeability increased, the ratio of clearance rates in the entire population showed a significant trend to increase in a non-linear fashion towards the value corresponding to the ratio of the free diffusion coefficients. We conclude that the diffusion of at least the larger of these two solutes through the pulmonary alveolar epithelium is restricted (i.e. associated with a reflection coefficient greater than zero). Cigarette smoking, however, does not appear to cause a loss of this restriction, and may increase solute clearance by other mechanisms, such as reducing fluid volume within the alveolus, thereby raising the local radiotracer concentration, or increasing the number of pores available for solute exchange without affecting pore size. Conversely, if restriction was lost in lung disease, the ratio of the clearance rates of two solutes of dissimilar sizes could be used to detect disease in smokers as well as non-smokers.

Anti-chelate antibodies after intraperitoneal yttrium-90-labeled monoclonal antibody immunoconjugates for ovarian cancer therapy.

UNLABELLED: The development of stable chelating agents for metal isotopes (e.g., 90Y) such as CITC-DTPA, a benzyl-analog of DTPA, allowed us to evaluate the efficacy of 90Y-labeled HMFG1 MAb administered intraperitoneally in patients with ovarian cancer. Our previous studies of 90Y-HMFG1 antibody, however, showed that all patients developed anti-chelate antibody responses (to the macrocycle benzyl-DOTA), resulting in clinical side effects in a significant percentage of this group. METHODS: We evaluated the immunogenicity of CITC-DTPA (administered to 12 patients as 90Y-HMFG1-CITC-DTPA after coupling it to HSA using solid-phase ELISA. RESULTS: Eleven of 12 evaluable patients developed anti-CITC-DTPA antibodies. Five patients (approximately 40%) developed hypersensitivity syndrome, most likely due to a type III immune reaction (serum sickness). Most patients had a low titer of pre-existing anti-chelate response which correlated positively with post-therapy response levels (p = 0.001). IgM anti-CITC-DTPA antibodies developed 2 wk while IgG antibodies developed 3 wk after treatment. Western blot analysis of post-therapy sera revealed a reaction with HSA-CITC-DTPA (60 kDa band) and no reaction with HSA or HSA-DTPA, whereas pre-therapy sera of the same patients were negative to all antigens. CONCLUSION: CITC-DTPA is immunogenic in patients after intraperitoneal administration of 90Y-CITC-DTPA labeled MAbs. Self-limiting clinical side effects consistent with a serum sickness-like immune reaction were observed in 5 of 12 patients.

Direct technetium-99m labeling of three anticancer monoclonal antibodies: stability, pharmacokinetics and imaging.

UNLABELLED: Monoclonal antibodies (MAbs) directly labeled with 99mTc have been used in a number of clinical immunoscintigraphic investigations. Three anti-cancer MAbs were radiolabeled with 99mTc using a reduction-mediated technique. The stability, biodistribution and in vivo pharmacokinetics were assessed and compared with the same antibodies labeled with 125I. METHODS: Immunoreactivity data were obtained by ELISA and RIA. Homogeneity and stability of radiolabeled antibodies (in vitro and in vivo) were measured by size-exclusion, fast protein liquid chromatography and SDS-PAGE. Pre-clinical, in vivo investigations utilized the nude mouse/HEp2 xenograft model, and clinical imaging and pharmacokinetic data were obtained from patients with confirmed or suspected lesions. RESULTS: Both 99mTc- and 125I-labeled antibodies were shown to be homogeneous and stable, although 99mTc-labeled antibody fragments were detected by SDS-PAGE. Pharmacokinetic studies in patients revealed a significant difference in the clearance rates between 99mTc- and 125I-labeled antibodies, with those labeled with 99mTc having a shorter biological half-life, indicating that the 99mTc-labeled antibodies may be less stable than the iodinated ones. Nevertheless, specific tumor localization was successfully demonstrated in nude mice bearing a human tumor xenograft using 125I- and 99mTc-labeled H17E2 antibody. Furthermore, in the clinic, using 99mTc-labeled HMFG1 and 1A3, successful imaging was achieved in 12 out of 19 patients with lesions for which these antibodies were specific. CONCLUSION: Anticancer MAbs radiolabelled using this reduction-mediated technique are suitable agents for clinical, immunoscintigraphic investigations.

90Y-labeled antibody uptake by human tumor xenografts and the effect of systemic administration of EDTA.

PURPOSE: A human tumor xenograft model was used to compare the tumor and normal tissue uptake of a tumor-associated monoclonal antibody radiolabeled with 125I or 90Y. METHODS AND MATERIALS: Nude mice bearing SC xenografts of the human colon adenocarcinoma, HT29, were injected with a mixture of 125I- and 90Y-DTPA-labeled AUA1 monoclonal antibody, which recognizes an antigen expressed on the surface of the tumor cells. In addition, the effect of systemic ethylenediaminetetraacetic acid (EDTA) administration on 90Y-labeled antibody clearance, tumor uptake of antibody and bone accumulation of 90Y was studied in a nude mouse model of intraperitoneal cancer. RESULTS: Both the absolute amount (%id.g-1) and the tumor:normal tissue ratios were superior for the 90Y-labeled antibody, compared with the iodinated antibody, with the notable exception of bone. These results suggest that 90Y is a preferable isotope to iodine for radioimmunotherapy of solid masses, but that myelotoxicity, due to bone uptake of released 90Y, will limit the radiation dose which can be given when DTPA is used to chelate the 90Y. The 90Y-labeled antibody showed similar serum stability in vitro in the presence or absence of EDTA after incubation for up to 48 h. In vivo, urine excretion of 90Y was significantly enhanced in mice receiving daily injections of 20 mg EDTA for 3 days, commencing 2 h after intraperitoneal antibody administration, compared with control mice. There was no significant difference in the tumor uptake of 90Y-labeled antibody in EDTA-treated and control mice at any time-point up to 9 days postinjection. However, the bone levels of 90Y were significantly reduced in EDTA-treated mice at all times from 1 to 9 days. CONCLUSION: Based on these results, it should be possible to increase the amount of 90Y-labeled antibody administered, by chelating the released 90Y with systemic EDTA to facilitate its excretion, without compromising tumor uptake of radiolabeled antibody.

Pharmacokinetics and toxicity of an yttrium-90-CITC-DTPA-HMFG1 radioimmunoconjugate for intraperitoneal radioimmunotherapy of ovarian cancer.

BACKGROUND: The intracavitary route for the administration of monoclonal antibodies is used in a variety of locally spreading cancers. The authors have been treating patients with ovarian cancer in Phase I and II studies assessing toxicity and response to improved radioimmunoconjugates. METHODS: Nineteen patients, 34-65 years of age, were treated with a new radioimmunoconjugate, 90Y-CITC-DTPA-HMFG1, instilled in the peritoneal cavity after second-look laparoscopy. Activity was increased in a stepwise fashion. RESULTS: Following the intraperitoneal administration of 90Y-CITC-DTPA-HMFG1, levels of the radioimmunoconjugate in the blood increased, reaching a peak of about 30% of injected activity at around 54 hours posttreatment. Approximately 18% of the radiolabel was excreted in the urine within 96 hours. Bone-marrow toxicity was the dose-limiting factor. Grade III platelet and granulocyte toxicity was observed at 19.3 mCi/m2. A type III immunologic response was observed in a number of patients. CONCLUSIONS: A dose of 18.5 mCi/m2 for subsequent treatments is recommended, based on a linear correlation of activity dose-to-body surface area. The clinical profile of a mild to moderate hypersensitivity syndrome is presented and hypotheses regarding its etiology are suggested.

Targeting of tumours with murine and reshaped human monoclonal antibodies against placental alkaline phosphatase: immunolocalisation, pharmacokinetics and immune response.

Anti-tumour monoclonal murine and humanised (reshaped human) antibodies (H17E2 and Hu2PLAP, respectively) against placental alkaline phosphatase (PLAP), radioactively labelled with indium-111 (111In) and iodine-123 (123I), were evaluated for their ability to localise mainly testicular and ovarian tumours in sequential pilot studies of the Hammersmith Oncology Group. 33 patients with active primary and/or metastatic testicular cancer were studied with the [111In]- or [123I]H17E2 antibody. 8 patients with testicular cancer were studied with the same antibody after being rendered free of disease after induction chemotherapy and surgical resection of residual tumour. 3 additional patients, 2 with ovarian cancer and 1 with testicular seminoma, were studied with [111In]H17E2 via a macrocyclic chelating agent (DOTA). 7 patients; 5 with ovarian cancer, 1 with breast cancer, and 1 with gastric cancer, received the reshaped human Hu2PLAP antibody [111In]DOTA labelled. One of these was imaged twice, with H17E2- and Hu2PLAP-DOTA-111In, respectively. In the initial 33 patients with active primary and/or metastatic testicular cancer, the presence of tumour was confirmed and correlated well with conventional radiological diagnostic methods, and in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging, but elevated serum tumour markers. In the 8 patients with complete remission (CR), imaging studies with the radiolabelled antibody did not show any localisation. The best images were obtained at 24 and 48 h after the [123I]- and [111In]H17E2, respectively. None of these patients developed human anti-mouse antibody responses (HAMA). Successful imaging with the reshaped human antibody, Hu2PLAP-DOTA-111In, was seen in 3 patients with PLAP-positive tumours (2 ovarian and 1 gastric cancer). The 3 negative patients were 1 in complete remission, 1 with PLAP-negative tumour and 1 who cleared the Hu2PLAP antibody immediately after infusion due to the presence of anti-chelating agent (anti-DOTA) antibodies from a previous H17E2-DOTA-111In scan. One patient with PLAP-negative breast carcinoma had a false-positive scan with Hu2PLAP, showing localisation to the pleural effusion. Antibody pharmacokinetics showed a mean t1/2 beta = 73.1 +/- 30.2 h (n = 5) for Hu2PLAP versus t1/2 beta = 27.2 +/- 5.9 h (n = 3) for H17E2 (P < 0.05). 2 patients receiving Hu2PLAP were excluded due to the rapid clearance of the radiolabel as a result of the presence of high HAMA and anti-chelate antibody levels, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvant therapy of ovarian cancer with radioactive monoclonal antibody.

Fifty-two patients with epithelial ovarian cancer were treated with yttrium-90-labelled monoclonal antibody HMFG1 administered intraperitoneally following conventional surgery and chemotherapy as part of an extended phase I-II trial. The treatment was well tolerated and the only significant toxicity observed was reversible myelosuppression as previously described. Following conventional surgery and chemotherapy, 21 out of the 52 patients had no evidence of residual disease and were regarded as receiving treatment in an adjuvant setting. To date, two of these patients have died of their disease (follow-up 3-62 months, median follow-up 35 months). This extended phase I-II study suggests that patients with advanced ovarian cancer who achieve a complete remission following conventional therapy may benefit from further treatment with intraperitoneal radioactive monoclonal antibody.

Technetium-99m-labeled antibodies. Stability compared to 125I labeled antibodies.

It is essential in any method for radiolabeling antibody with 99mTc that the labeling procedure is rapid and reliable, producing a highly stable 99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (99mTc and 125I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (99mTc or 125I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of 99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr < IgG) that were not detected by the same analysis of 125I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the 99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of 99mTc-labeled material with a Mr < IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the 99mTc equivalent corresponds to free iodide.

Development of humoral immune responses against a macrocyclic chelating agent (DOTA) in cancer patients receiving radioimmunoconjugates for imaging and therapy.

The development of stable immunoconjugates by the advent of macrocyclic metal chelating agents (DOTA) has enabled us to study the ability of 111In-DOTA-labeled monoclonal antibodies to detect tumor lesions in a pilot radioimmunolocalization study, as well as to evaluate the kinetics, toxicity, and efficacy of i.p. administered 90Y-DOTA-labeled murine monoclonal antibody in a Phase I/II clinical trial of advanced ovarian cancer. The development of serum sickness-like reactions in three of six treated patients, in the absence of previous monoclonal antibody administration, led us to study the potential immunogenicity of the new chelate. Six patients with ovarian cancer received 25 mg of HMFG1 monoclonal antibody coupled with 90Y-DOTA (doses of radioactivity, 15 to 25 mCi), administered i.p. Eight patients with various malignant tumors received low doses (220 micrograms to 1 mg) of monoclonal antibodies, labeled with 111In-DOTA, i.v. for imaging studies. Using a solid-phase enzyme-linked immunosorbent assay method, the immunogenicity of DOTA was evaluated. Serial dilutions of patients' sera, before and after imaging or therapy with DOTA-coupled monoclonal antibodies, as well as sera from patients who did not receive DOTA-coupled antibody, were screened on enzyme-linked immunosorbent assay plates coated with human serum albumin (HSA), HSA-2-iminothiolane, and HSA-2-iminothiolane-benzyl-DOTA. All patients treated with i.p. monoclonal antibody developed anti-DOTA antibodies. Four of eight patients who received i.v. "imaging" doses of DOTA-coupled monoclonal antibody developed antibodies against DOTA. The levels of anti-DOTA response correlated with the amount of injected radioimmunoconjugate (r = 0.889, P less than 0.001). None of the patients who received DOTA-coupled antibody had detectable antibodies against the macrocycle before immunoconjugate administration. We then addressed further the restriction of the immune response against the macrocycle. We found that there was no or very low response against the aromatic ring attached to DOTA. Most, if not all, of the immune response is directed against the DOTA ring structure. Affinity purification of anti-DOTA antibody from serum enabled quantitation of these antibodies in the serum of patients. An inverse, statistically significant correlation was observed between the percentage of binding inhibition of a patient's serum to DOTA, by HSA-2-iminothiolane-DOTA (100 micrograms/ml) and the level of anti-DOTA immunoglobulin in the serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumour localisation with a radioactively labelled reshaped human monoclonal antibody.

A genetically reshaped human IgG1 monoclonal antibody (Hu2PLAP) with anti-tumour specificity, was radiolabelled with Indium-111 by chelation with a new macrocyclic compound (DOTA) which allows the production of stable radioimmunoconjugates for in vivo application. This was used to image seven patients with malignant disease, of whom two had been previously exposed to mouse monoclonal antibodies and had developed human anti-mouse antibodies (HAMA). Successful tumour localisation was seen in the four patients with active disease and antigen positive tumours. No patient showed any antibody responses against Hu2PLAP, but three out of six patients tested showed an immune response against the macrocycle DOTA. Reshaped human monoclonal antibodies with anti-tumour specificity may facilitate repeated administrations of radioactive antibodies, thus allowing new possibilities, both in the diagnosis and treatment of cancer.

Quantitative measurement of monoclonal antibody distribution and blood flow using positron emission tomography and 124iodine in patients with breast cancer.

The uptake and in vivo quantitation of monoclonal antibodies (MAbs) has been measured non-invasively using positron emission tomography (PET) and 124iodine in 9 patients with breast ductal carcinoma. Blood-flow measurements were also made using 15oxygen-labelled water and PET to evaluate antibody delivery; 7 patients were studied with HMFGI antibody and 2 patients with a non-specific antibody. Tumour uptake ranged from 2-7.7 x 10(-3)% of injected dose per gram of tissue. Values for normal tissues including liver, lung and bone were also obtained. In 2 out of 7 patients studied with the specific antibody, uptake was greater than that seen with the non-specific antibody. There was no correlation between antibody uptake and blood flow. This report exemplifies the potential of PET for the non-invasive and accurate quantitative assessment of targeted antibody which is a prerequisite to therapy.

Intraperitoneal yttrium-90-labeled monoclonal antibody in ovarian cancer.

From March 1987 to March 1988, a phase I to II study was carried out in 25 patients with ovarian cancer. They received escalating doses of intraperitoneally (IP) administered yttrium-90 (Y-90)-labeled monoclonal antibody, HMFG1, against a tumor cell-surface antigen. Myelosuppression prevented an escalation of the administered Y-90 activity above 25 mCi. Y-90-labeled antibody was absorbed from the peritoneal cavity into the circulation. Maximum blood Y-90 activity was observed 40 hours after the IP injection with a mean of 21% of the injected activity (range, 14.2% to 26.4%) in the circulation. The radiation dose the bone marrow received from circulating Y-90-labeled antibody (the blood radiation dose) was calculated by applying the Medical Internal Radiation Dose (MIRD) formulation to the measured Y-90 activity in patients blood. Myelosuppression occurred following calculated blood radiation doses to bone marrow of only 10 to 30 cGy. The excessive myelosuppression following such modest radiation doses from circulating Y-90-labeled antibody could be explained by the uptake of Y-90 by bone. In an attempt to reduce bone absorption of Y-90, seven patients received an intravenous (IV) infusion of EDTA (Sinclair Pharmaceuticals Ltd, Godalming, United Kingdom). This increased the urinary excretion of Y-90 from a mean of 11.1% to 32.3% of the injected activity (P = .0001). Fourteen patients had assessable tumor at laparoscopy. Tumor regression was observed in one patient, and palliation of ascites in a further patient.

Imaging of tumor in patients with indium-111-labeled biotin and streptavidin-conjugated antibodies: preliminary communication.

Tumor localization in patients has been achieved through the in vivo use of streptavidin and biotin. In these preliminary studies, the monoclonal antibody HMFG1 was conjugated with streptavidin and 1 mg was administered intravenously to each of 10 patients with documented squamous cell carcinoma of the lung. Two to 3 days later, 111In-labeled biotin was also administered intravenously. No evidence of toxicity was observed. Background radioactivity levels were reduced in liver (1% ID at 24 hr) and kidneys (2%) and in all other normal tissues and blood. Images of lung tumor were obtained in as little as 2 hr following administration of labeled biotin. In eight patients, tumor was detected with labeled biotin alone without the previous administration of streptavidin-conjugated antibody but in three of these patients, the images were improved with the prior administration of conjugated antibody. These results suggest that this approach may improve the tumor-to-normal tissue radioactivity ratios in radioimmunotargeting.

Immunolocalisation of testicular tumours using radiolabelled monoclonal antibody to placental alkaline phosphatase.

Tumour associated monoclonal antibody against placental alkaline phosphatase (H17E2) was radiolabelled with Indium-111 and Iodine-123 and administered intravenously in 33 patients with primary and/or metastatic testicular tumour, as well as in 8 patients who were in complete remission after surgical excision of the tumour. The presence of a tumour was confirmed and correlated well with conventional diagnostic techniques and, in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging and with elevated serum markers. In addition, in one patient the CT produced a false positive result where the antibody scan was negative. Finally, the absence of tumour was confirmed in all 8 cases of patients in complete remission. All patients studied with Indium-labelled antibody had observable concentrations of the radiolabel in the liver (estimated to be approximately 30% of the administered dose), as well as in the kidneys and spleen. The patients studied with the Iodine-123 labelled antibody had observable concentrations in the thyroid gland and the stomach. The best images were seen at 48 and 24 hrs after the Indium and Iodine radiolabelled antibody respectively. No human anti-mouse antibody was detected in any of our patients, even in those who received 2 and 3 administrations, with the highest amount of administered protein being 800 micrograms. No toxicity was encountered in any of our patients in 4 months of follow-up. This method may be of clinical value in patients with testicular neoplasma and represents a new addition to current imaging techniques. A positive scan indicates the definite presence of a tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrocyclic chelates of radiometals for diagnosis and therapy.

Monoclonal antibody technology allows the specificity of an antibody for its antigen to be used in targeting cancer cells. Stable attachment of metal ions to antibodies by means of 'bifunctional' chelating agents can add the nuclear, physical and chemical properties of the metallic elements to these target-selective proteins. The conjugation of metals--particularly radionuclides--to monoclonal antibodies results in agents for radioimmunotherapy and other medical applications. Chelators that can hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to non-target cells. Derivatives of polyazamacrocycles (bearing a C-substituted functional group for antibody attachment) can exhibit remarkable kinetic inertness. We have developed a new synthetic route these macrocycles via peptide synthesis and intramolecular tosylamide ring closure. Incubation of the yttrium complex of 2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N1N",N'"-te traacetic acid (nitrobenzyl-DOTA) for 18 days in serum results in loss of so little yttrium from the complex (less than 0.5%) that the rate of loss cannot be measured under these conditions. In animal models, conjugates of this chelate with monoclonal antibodies show much lower levels of yttrium in the bone than are found with DTPA chelates prepared from the cyclic anhydride. The rates of loss of indium and cobalt from nitrobenzyl-DOTA in serum are slower than from previously studied chelates. Preliminary clinical imaging studies of 111In-labeled monoclonal antibody conjugates of DOTA show highly encouraging results.

Intraperitoneally administered 90Y-labelled monoclonal antibodies as a third line of treatment in ovarian cancer. A phase 1-2 trial: problems encountered and possible solutions.

A phase 1-2 trial of 90Y-labelled monoclonal antibody, HMFG1 administered intraperitoneally to 30 patients with ovarian carcinoma is presented. The problems encountered with myelotoxicity are described, and the steps which have so far been taken to overcome this and to increase the dose of 90Y to an estimated tumouricidal level. Bone deposition of free 90Y limits the dose which can be administered without severe bone marrow toxicity. The intravenous use of a chelating agent, Ledclair (EDTA) has allowed the dose to be increased from 18 to 30 mCi without causing severe myelotoxicity. 90Y-DTPA MAb is an unstable immunoconjugate in vivo and in vitro. The use of a more stable linkage such as a macrocycle should enable the administered dose to be increased without increasing the amount of free 90Y which becomes available to be deposited in bone. This would be expected to reduce toxicity and increase therapeutic efficacy.

Preparation and in vivo study of 124I-labelled monoclonal antibody H17E2 in a human tumour xenograft model. A prelude to positron emission tomography (PET).

Positron emission tomography (PET) is a powerful nuclear medicine technique which, unlike conventional gamma camera tomography, relies on the coincidental detection of the two 511 keV gamma photons produced from the annihilation of a single positron. Hence good spatial resolution and accurate quantitation may be achieved. 124I (t1/2 = 4 days), a positron-emitting isotope of iodine, was chosen for our initial PET studies because the techniques of antibody radio-iodination are well established. The murine monoclonal antibody H17E2 detecting placental alkaline phosphatase (PLAP) was radiolabelled using the Iodogen method. A specific activity of 2.3 microCi microgram-1 was achieved with a radiolabelling efficiency of 91%. Nude mice bearing subcutaneous HEp2 human tumour xenografts (a PLAP expressing cell-line) received 8.3 micrograms (18.8 microCi) of H17E2-124I by intraperitoneal injection. Animals were killed and dissected at 5 h, 1, 2, 3, and 7 days, and radioactivity was assessed in tumour and normal tissues. The half-life in the blood of H17E2-124I was 132 h as compared with 141 h for H17E2-131I. Activity in tumour rose to 4.26% injected dose g-1 by 48 h and remained at this level until day 7, giving a tumour:blood ratio of 0.78 at this time. The percentage injected dose g-1 in all tissues (with the exception of tumour) decreased with time giving tumour:tissue ratios greater than 1.00 from 24 h onwards in all cases except blood. In conclusion, tumour localization of H17E2-124I has been successfully achieved in this animal model. This demonstrates the feasibility of using tumour-associated monoclonal antibodies radiolabelled with a positron-emitting isotope for tumour localization studies.

Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase.

Twenty-seven patients with brain glioma were scanned using 123I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.