In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody (99m)Tc-D10 for visualizing small tumour lesions with volumes below 100 mm(3) by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody (99m)Tc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm(3) ± 21.2 and 26.6 mm(3) ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of (99m)Tc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody (99m)Tc-D10.

Preparation and Evaluation of 99mTc-labeled anti-CD11b Antibody Targeting Inflammatory Microenvironment for Colon Cancer Imaging.

CD11b, an active constituent of innate immune response highly expressed in myeloid-derived suppressor cells (MDSCs), can be used as a marker of inflammatory microenvironment, particularly in tumor tissues. In this research, we aimed to fabricate a (99m)Tc-labeled anti-CD11b antibody as a probe for CD11b(+) myeloid cells in colon cancer imaging with single-photon emission computed tomography (SPECT). In situ murine colon tumor model was established in histidine decarboxylase knockout (Hdc(-/-)) mice by chemicals induction. (99m)Tc-labeled anti-CD11b was obtained with labeling yields of over 30% and radiochemical purity of over 95%. Micro-SPECT/CT scans were performed at 6 h post injection to investigate biodistributions and targeting of the probe. In situ colonic neoplasma as small as 3 mm diameters was clearly identified by imaging; after dissection of the animal, anti-CD11b immunofluorescence staining was performed to identify infiltration of CD11b+ MDSCs in microenvironment of colonic neoplasms. In addition, the images displayed intense signal from bone marrow and spleen, which indicated the origin and migration of CD11b(+) MDSCs in vivo, and these results were further proved by flow cytometry analysis. Therefore, (99m)Tc-labeled anti-CD11b SPECT displayed the potential to facilitate the diagnosis of colon tumor in very early stage via detection of inflammatory microenvironment.

Molecular pathway-specific 99mTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid liver imaging to assess innate immune responses induced by cell transplantation.

OBJECTIVES: Inflammatory responses after cell transplantation impair engraftment of transplanted cells. We studied whether perturbations in specific molecular pathways after inflammation in a syngeneic cell transplantation model could be identified by noninvasive imaging. METHODS: After transplanting hepatocytes into the liver of dipeptidyl peptidase IV-deficient Fischer 344 rats, we imaged hepatobiliary excretion of ppmTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid (99mTc-mebrofenin). Fractional retention of peak hepatic mebrofenin activity over 60-min periods was correlated with parameters of hepatic inflammation. RESULTS: In healthy animals, 28+/-6% 99mTc-mebrofenin activity was in the liver after 60 min, whereas cell transplantation dose-dependently inhibited excretion of 99mTc-mebrofenin, P value of less than 0.001. Resolution of this abnormality in 99mTc-mebrofenin transport required 2 weeks in the setting of prolonged activation of Kupffer cells with increased TNF-alpha and IL-6 expression. Hepatic transport of 00mTc-mebrofenin was promptly restored by anti-inflammatory treatments, including inhibition of cyclooxygenase activity, depletion of neutrophils, or blocking of inflammatory cytokines before cell transplantation. Moreover, these treatments improved transplanted cell engraftment. CONCLUSION: Molecular pathway-based imaging offers appropriate noninvasive means to address activation of innate immune responses. This will help in developing suitable strategies for characterizing and overcoming immune responses for cell and gene therapy.

99mTc-labelled rituximab, a new non-Hodgkin's lymphoma imaging agent: first clinical experience.

OBJECTIVE: This study was performed to explore the possibility of using Tc-rituximab as an imaging agent to assess expression of CD20 antigen in patients with B-cell non-Hodgkin's lymphoma (NHL) before (radio) immunotherapy, for staging and subsequent evaluation of remission of NHL. METHODS: Rituximab was purified from Mabthera and photoactivated by ultraviolet light. The irradiated solution was aliquoted and labelled with pertechnetate. The effectiveness of the labelling method was evaluated by determination of the number of free thiol groups per photoreduced antibody, radiochemical purity determination and in-vitro stability. Immunoreactivity of Tc-rituximab was assessed on Ramos cells using a direct binding assay. Ten patients (age 31-70 years, mean 50 years) were included, nine with CD20 B-cell NHL and one with CD20-NHL. Whole-body and single photon emission computed tomography images were taken 1, 3, 6 and 20 h postinjection of Tc-rituximab. Scintigraphic results were compared with computerized tomography (CT) findings. RESULTS: In all cases radiochemical purity over 95% was observed with preserved affinity for CD20 antigen. In all patients expected activity was seen in the blood pool, liver, kidneys and spleen. Pathological, moderately to markedly increased Tc-rituximab activity was seen in all but one CT-confirmed NHL involved sites 6 and 20 h postinjection. In one patient, increased activity of Tc-rituximab was additionally seen in one region not seen on CT. In three patients increased accumulation was seen in bone marrow. CONCLUSION: Tc-rituximab is a promising imaging agent suitable for assessing expression of CD20 in patients with NHL before (radio) immunotherapy.

Radioimmuno targetting (99m)technetium labeled anti-epidermal growth factor receptor monoclonal antibodies in experimental tumor models.

AIM: Monoclonal antibodies (MAb) directed at the extra cellular domain (ECD) of epidermal growth factor receptor (EGFR) offer a promising strategy for diagnosis and therapy of cancers that over-express EGFR. Radiolabelled MAbs against cell surface antigens have improved in vivo tumor diagnosis and treatment. EGFR over-expression has been reported in a wide range of carcinomas especially of the head and neck, breast, etc., and is associated with poor prognosis and resistance to therapy. CIBCNSH3 is a murine MAb generated to the ECD of EGFR in our laboratory and has been extensively characterized and has proven antitumor activity. The tumor targeting potential of (99m)Tc labelled CIBCNSH3 in an experimental tumor model is discussed in this paper. METHODS: A431, an epidermoid carcinoma cell line with overexpression of EGFR, SUDHLH, a lymphoma cell line was used to induce xenografts in inbred adult female BALB/C mice and used for the study. A reduction mediated method of (99m)Tc labelling was adopted to label the MAb. Scintiscan pictures were taken at different time intervals after i.v. administration of the (99m)Tc labelled MAb using a gamma camera and results were correlated with those of biodistribution studies. RESULTS: Immunoscan pictures taken at different time periods showed high uptake of the radioimmunoconjugate by the tumor providing clear tumor images and no uptake in control animals with lymphoma xenografts. Results of scan pictures correlated well with the biodistribution studies. CONCLUSION: The radioimmunoconjugate (99m)Tc-CIBCNSH3 appears to be a promising tool in identifying any early recurrence and micro-metastasis of lesions that overexpress EGFR.

Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide.

The aim of this study was to localize (99m)Tc and (188)Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 x 734 (Fab' x Fab'), anti-carcinoembryonic antigen [CEA] x anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N'-o-Phenylenedimaleimide was used to cross-link the Fab' fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH(2) ¿TscG = 3-thiosemicarbazonylglyoxyl¿], with two indium-DTPAs and a chelate for selectively binding (99m)Tc or (188)Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with (99m)Tc (94-99%) at up to 1836 Ci/mmol and with (188)Re (97%) at 459-945 Ci/mmol of peptide. [(99m)Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 x murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) x 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 x m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 x m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 microg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 x 734, respectively), [(188)Re]IMP-192 (7 microCi) or [(99m)Tc]IMP-192 (10 microCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [(99m)Tc] or [(188)Re]IMP-192 was 12.6 +/- 5.2 and 16.9 +/- 5.5% ID/g at 3 h postinjection, respectively. Tumor/nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with (99m)Tc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when (188)Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.

A new method of technetium-99m labeling of monoclonal antibodies through sugar residues. A study with TAG-72 specific CC-49 antibody.

We have developed a very efficient labeling technique for monoclonal antibodies with technetium-99m. Oxidation of sugar residues on the IgG class of antibodies leads to the generation of aldehyde groups which are further reacted with two newly developed hydrazide compounds. This methodology introduces sulfhydryl groups on the antibody through sugar residues which can be labeled with technetium-99m. We have studied the TAG-72 specific second generation antibody CC-49. The specific activity of the labeled antibody was high without loss of its immunoreactivity.

Rapid infarct imaging with a technetium-99m-labeled antimyosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction.

Studies of monoclonal antibody-based imaging agents show that blood clearance is inversely proportional to molecular size, i.e., Fab or Fab' > F(ab')2 > IgG. Indium-111-antimyosin Fab-DTPA is a highly specific and sensitive marker for myocardial necrosis. An improvement on current antibody diagnostic imaging may result from the use of smaller labeled fragments. We report the first in vivo targeting of acute myocardial infarction with a novel recombinant single-chain Fv (sFv) antimyosin protein. The sFv (MW = 27,594) is approximately one-half the size of the Fab and is comprised of the heavy and light chain variable regions from the myosin-specific murine monoclonal antibody R11D10 which were joined by a 15-amino-acid linker and expressed as a fusion protein (sFv) in E. coli. The binding affinity of the sFv for cardiac myosin was similar to the affinity observed for the Fab fragment. Technetium-99m labeling of the sFv was accomplished by the attachment of a cleavable, ester-linked bifunctional chelator (RP-1). Comparative studies in mice showed 99mTc-sFv-RP-1 cleared significantly faster (p < 0.001) than 99mTc-Fab'-RP-1 and 111In-Fab-DTPA antimyosin fragments. Furthermore, measurement of 99mTc-sFv-RP-1 blood clearance in a canine model of acute myocardial infarction gave a mean T1/2 of 0.54 +/- 0.13 hr versus 2.80 +/- 0.57 and 2.58 +/- 0.64 hr for Fab-DTPA and Fab'-RP-1 (p < 0.05), respectively. Despite its comparatively rapid clearance, 99mTc sFv-RP-1 had similar uptake in the infarct compared to the Fab'-RP-1. In addition, infarct visualization was more rapid with the sFv. Thus, these data demonstrate antimyosin sFv possesses characteristics necessary for rapid imaging of myocardial necrosis.