Antibody guided targeting of non-small cell lung cancer using 111In-labeled HMFG1 F(ab')2 fragments.

Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111In. Quality control studies showed efficient incorporation of 111In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody. Based on these findings, we recommend that in order to demonstrate specific radioimmunolocalization, patients with lung and possibly other tumor types should be studied using both specific and nonspecific antibodies.

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)

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.

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.

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.

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.