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Blood clearance () and urinary (OE) and fecal (F) excretion of 123 I-mAb 14C5 (%ID) in mice after intravenous injection. Each time point represents mean SD of 3 animals. Corresponding curve fittings, as determined by SPSS 10.0 program, are indicated by solid lines.
Source publication
This study reports on the in vitro evaluation, biodistribution, and dosimetry of (123)I-labeled monoclonal antibody (mAb) 14C5, a new antibody-based agent proposed for radioimmunodetection of tumor growth and metastasis in vivo.
(123)I-mAb 14C5 was prepared by direct iodination and tested for stability in vitro. Binding assays were performed on hum...
Contexts in source publication
Context 1
... shown in Figure 3, 123 I-mAb 14C5 was slowly cleared from the blood (9.7 1.4 %ID remaining at 48 h after injection) following a biexponential pharmacokinetic be- havior. Fitting of the blood clearance curve was performed with the SPSS 10.0 software package, using the method of least squares, to calculate the biologic half-life (t 1/2 ) of the tracer (r 2 0.95). The biexponential blood clearance of 123 I-mAb 14C5 was characterized by a fast phase (t 1/2, 1.43 0.42 h, 62.8% of total circulating radioactivity in the (13,14,25). ‡ Values represent percentage of specific 123 I-mAb 14C5 binding to each carcinoma cell type (total bound minus nonspecifically bound) as determined by Equation 1 (n 6). blood) and a slow phase (t 1/2, 130.4 100.0 h, 37.2%). The remaining radioactivity (68 %ID) was rapidly distrib- uted into the extravascular compartments in the initial phase of the tracer biodistribution. Kinetic parameters for blood clearance are presented in Table 2. Blood-pool activity was at any time substantially higher than tracer uptake in any of the other investigated organs. 123 I-mAb 14C5 did not cross the blood-brain barrier as indicated by the low brain uptake (1.0 %ID/g on average). The biodistribution data are presented in Table 3. The highest accumulation of radioac- tivity occurred in lungs (20.4 3.7 %ID/g), liver (15.1 1.4 %ID/g), and kidneys (11.1 1.1 %ID/g) within 5 min after injection and to a lesser extent in the spleen (9.2 2.4 %ID/g) at 20 min after injection. A delayed uptake was observed in stomach (12.8% 2.4 %ID/g) and urinary bladder (8.7 5.2 %ID/g) at 3 and 6 h after injection, respectively (Table 3). 123 I-mAb 14C5 was mainly cleared by kidneys, with 40.7 2.9 %ID being excreted in urine at 48 h after injection, representing 90.8% of the total excreted radioactivity. Fecal excretion 48 h after tracer injection (4.1 1.0 %ID) contributed only 9.2% to the total excreted activity as depicted in Figure ...
Context 2
... shown in Figure 3, 123 I-mAb 14C5 was slowly cleared from the blood (9.7 1.4 %ID remaining at 48 h after injection) following a biexponential pharmacokinetic be- havior. Fitting of the blood clearance curve was performed with the SPSS 10.0 software package, using the method of least squares, to calculate the biologic half-life (t 1/2 ) of the tracer (r 2 0.95). The biexponential blood clearance of 123 I-mAb 14C5 was characterized by a fast phase (t 1/2, 1.43 0.42 h, 62.8% of total circulating radioactivity in the (13,14,25). ‡ Values represent percentage of specific 123 I-mAb 14C5 binding to each carcinoma cell type (total bound minus nonspecifically bound) as determined by Equation 1 (n 6). blood) and a slow phase (t 1/2, 130.4 100.0 h, 37.2%). The remaining radioactivity (68 %ID) was rapidly distrib- uted into the extravascular compartments in the initial phase of the tracer biodistribution. Kinetic parameters for blood clearance are presented in Table 2. Blood-pool activity was at any time substantially higher than tracer uptake in any of the other investigated organs. 123 I-mAb 14C5 did not cross the blood-brain barrier as indicated by the low brain uptake (1.0 %ID/g on average). The biodistribution data are presented in Table 3. The highest accumulation of radioac- tivity occurred in lungs (20.4 3.7 %ID/g), liver (15.1 1.4 %ID/g), and kidneys (11.1 1.1 %ID/g) within 5 min after injection and to a lesser extent in the spleen (9.2 2.4 %ID/g) at 20 min after injection. A delayed uptake was observed in stomach (12.8% 2.4 %ID/g) and urinary bladder (8.7 5.2 %ID/g) at 3 and 6 h after injection, respectively (Table 3). 123 I-mAb 14C5 was mainly cleared by kidneys, with 40.7 2.9 %ID being excreted in urine at 48 h after injection, representing 90.8% of the total excreted radioactivity. Fecal excretion 48 h after tracer injection (4.1 1.0 %ID) contributed only 9.2% to the total excreted activity as depicted in Figure ...
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Citations
... The murine mAb 14C5 binds the integrin α v β 5 , a close relative of α v β 3. In vitro and in vivo studies showed the radioiodinated murine mAb 14C5 to have promising properties for diagnostic and therapeutic applications against integrin α v β 5-expressing tumor cells and/or tumor surrounding stromal cells, for example, fibroblasts[11][12][13][14]. Full-sized Abs need to overcome some obstacles before penetrating into a tumor. ...
Background
Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvβ5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics.
Methods
In vitro stability, specificity, and affinity of radioiodinated chAb and fragments (Iodo-Gen method) were examined on high-expressing αvβ5 A549 lung tumor cells. In vivo biodistribution and pharmacokinetic characteristics were studied in A549 lung tumor-bearing Swiss Nu/Nu mice.
Results
Saturation binding experiments revealed high in vitro affinity of radioiodinated chAb, F(ab')2, and Fab, with dissociation constants (KD) of 1.19 ± 0.19, 0.68 ± 0.10, and 2.11 ± 0.58 nM, respectively. ChAb 14C5 showed highest tumor uptake (approximately 10%ID/g) at 24 h post injection, corresponding with other high-affinity Abs. ChF(ab')2 and chFab fragments showed faster clearance from the blood compared to the intact Ab.
Conclusions
The chimerization of mAb 14C5 and its fragments has no or negligible effect on the properties of the antibody. In vitro and in vivo properties show that the chAb 14C5 is promising for radioimmunotherapy, due to its high maximum tumor uptake and its long retention in the tumor. The chF(ab')2 fragment shows a similar receptor affinity and a faster blood clearance, causing less non-specific retention than the chAb. Due to their fast blood clearance, the fragments show high potential for radioimmunodiagnosis.
... Recently, a promising new antibody targeting the integrin a v ß 5 was identifiedfor diagnosing human breast, colon, squamous-cell, and lung cancers. [10][11][12][13][14][15] By FACScan analysis, the integrin a v ß 5 was shown to be overexpressed on the tumor surfaces of several cancer types, including pancreatic cancer. 13,15 Immunohistologic staining of human lung, colon, and breast tumor tissues with mAb 14C5 showed antigen expression predominantly in the stroma surrounding the tumor cells and on the stromal fibroblasts (squamous-cell carcinoma, 5 of 5; lung adenocarcinoma, 3 of 3; large cell carcinoma, 1 of 1; colon adenocarcinoma, 19 of 20) and was only localized on the tumor cells themselves in some cases (squamous-cell carcinoma, 2 of 5; lung adenocarcinoma, 1 of 3; large-cell carcinoma, 0 of 1; colon adenocarcinoma 10 of 20). ...
... Iodination of mAbs 14C5 and MAB002 was performed using the Iodo-Gen method as described previously. 10,13,14 Preparation of 111 In-labeled 14C5. The bifunctional chelators p-SCN-Bz-DTPA and p-SCN-Bz-DOTA were purchased from Macrocyclics. ...
The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.
... 123 I-labeled mAb 14C5 could be used for tumour diagnosis or to evaluate chemotherapy response (RID), whereas 131 I-labeled mAb 14C5 could be used to ablate primary tumour cells, metastatic cells and possibly cancer-associated fibroblasts (RIT). Lahorte et al.(2004) reported on the radioiodination of mAb 14C5 with 123 I. ...
In search of new antibody therapeutics for inhibition of metastatic cancer, the foci of this research were to identify and further characterize antigen 14C5 as potential target for monoclonal antibody 14C5 (mAb 14C5)-based imunotherapies and to explore the targeting properties of radioiodinated mAb 14C5 for radioimmunodetection and –therapy. In the first part of the thesis we show that antigen 14C5 is the αvβ5 integrin receptor. Immunocytochemical analysis and flow cytometry analysis show that the expression of antigen 14C5 is present in varying degrees on neoplastic human cell lines from different origins. Antigen 14C5 expression on lung and colon carcinoma tissues mainly occurred in the stroma and on stromal fibroblasts surrounding the tumour cells and to a lesser extent at the tumour cells. Further studies on the expression of antigen 14C5 have to show whether antigen 14C5 is an indicator of the fibroblast cancer-associated phenotype. The second part of the thesis explored the potential of radioiodinated mAb 14C5 as agent for radioimmunodetection and –therapy. Radioiodinated mAb 14C5 binds its antigen with high affinity, shows good and specific tumour uptake in vivo and good planar gamma camera imaging quality. However, the blood clearance is slow. Therefore, Fab and F(ab’)2 fragments of intact mAb 14C5 were generated. Radioiodinated F(ab’)2 still binds antigen 14C5 with high affinity, while radioiodinated Fab looses affinity. Fragments showed increased tumour-to-blood and tumour-to-muscle ratios, but the overall tumour uptake of both fragments was significantly reduced. Internalization can lead to a rapid loss of the radiolabel from the tumour cell. We investigated the fate of the 125I-labelled mAb 14C5-antigen complex. Studies were also conducted with 125I-labeled F(ab’)2 and Fab fragments. Genetic engineering provides powerful tools for manipulating the structure and pharmacokinetic properties of antibodies in order to obtain optimal targeting properties. In this context, the variable heavy and variable light chain genes of mAb 14C5 were sequenced and the complement determining regions characterizing mAb 14C5 were identified. This opens the way to a broad range of applications including altering pharmacokinetics, reducing immunogenicity, the use of a variety of isotopes, conjugation of drugs or toxins, the design of bispecific antibodies and pretargeting strategies.
... The [ 123 I]AD-AM EDE for an adult male, calculated using attenuationcorrected images, was 3.37Â10 À2 mSv/MBq. Based on the average EDE value derived from this study, subjects would receive an EDE of approximately 6.23 mSv using a general injection dose of 185 MBq [ 123 I]ADAM, which is comparable to the average EDE per patient in other 123 I studies using standard nuclear medicine procedures [29][30][31][32]. However, redistribution of the tracer from extracerebral compartments (such as the lung) to plasma and brain must be carefully taken into consideration while quantifying the SB in the brain. ...
[(123)I]-2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine ([(123)I]ADAM), a novel radiotracer, has promising application in the imaging of the serotonin transporter (SERT) in the human brain. In this study, the optimal scanning time for acquiring brain single photon emission computed tomography (SPECT) images was determined by performing dynamic SPECT studies at intervals from 0 to 6 h postinjection of [(123)I]ADAM. Additionally, radiation-absorbed doses were determined for three healthy human subjects using attenuation-corrected images.
Twelve subjects were randomized into one of three study groups as follows: whole-body distribution imaging (n=3), dynamic SPECT imaging (n=3) and brain SPECT imaging (n=6). The radiation-absorbed dose was calculated using MIRDOSE 3.0 software with attenuation-corrected data. The specific binding (SB) ratio of the brain stem was measured from dynamic SPECT images to determine the optimal scanning time.
Dynamic SPECT images showed that the SB of the brain stem gradually increased to a maximum 4 h postinjection. Single photon emission computed tomography images at 4 h postinjection showed a high uptake of the radiotracer (SB) in the hypothalamus (1.40+/-0.12), brain stem (1.44+/-0.16), pons (1.13+/-0.14) and medial temporal lobe (0.59+/-0.10). The mean adult male value of effective dose was 3.37 x 10(-2) mSv/MBq with a 4.8-h urine-voiding interval. Initial high uptake in SERT-rich sites was demonstrated in the lung and brain. A prominent washout of the radiotracer from the lung further increased brain radioactivity that reached a peak value of 5.03% of injected dose 40 min postinjection.
[(123)I]ADAM is a promising radiotracer for SPECT imaging of SERT in humans with acceptable dosimetry and high uptake in SERT-rich regions. Brain SPECT images taken within 4 h following injection show optimal levels of radiotracer uptake in known SERT sites. However, dynamic changes in lung SERT distribution must be carefully evaluated.
In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts.
This study identifies and characterizes the antigen recognized by monoclonal antibody (mAb) 14C5. We compared the expression of antigen 14C5 with the expression of eight integrin subunits (alpha1, alpha2, alpha3, alphav, beta1, beta2, beta3, and beta4) and three integrin heterodimers (alphavbeta3, alphavbeta5, and alpha5beta1) by flow cytometry. Antigen 14C5 showed a similar expression to alphavbeta5 in eight different epithelial cancer cell lines (A549, A2058, C32, Capan-2, Colo16, HT-1080, HT-29, and SKBR-3). Specific binding of P1F6, an anti-alphavbeta5 specific antibody, was blocked by mAb 14C5. After transient expression of alphavbeta5 in 14C5-negative Colo16 cells, mAb 14C5 was able to bind a subpopulation of alphavbeta5-positive cells. We evaluated the tissue distribution of the 14C5 antigen in colon (n = 20) and lung (n = 16) cancer tissues. The colon carcinoma cells stained positive for 14C5 in 50% of tumors analyzed, whereas bronchoalveolar lung carcinoma and typical carcinoid were not positive for the antigen. More common types of non-small cell lung cancer, i.e., squamous (n = 5) and adenocarcinoma (n = 3), stained positive in 2 of 5 squamous carcinomas and in 1 of 3 investigated adenocarcinoma. Colon (95%) and lung (50%) carcinoma tissues showed extensive expression of antigen 14C5 in the stroma surrounding the tumor cells and on the membrane of the adjacent fibroblasts. We show for the first time that mAb 14C5 binds the vascular integrin alphavbeta5, suggesting that mAb 14C5 can be used as a screening agent to select colon and lung cancer patients that are eligible for anti-alphavbeta5-based therapies.
The monoclonal antibody (mAb) 14C5 is a murine IgG1 directed against a yet undefined molecule involved in cell substrate adhesion found on the surface of malignant breast cancer tissue. mAb 14C5 is able to inhibit cell substrate adhesion and invasion of breast cancer cells in vitro. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast, no expression of the antigen 14C5 occurs. The aim of this study was to investigate the in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 as a novel agent for radioimmunodetection and radioimmunotherapy.
Internalization of mAb 14C5 was investigated with 125I-labeled mAb 14C5 and by confocal laser scanning microscopy. Biodistribution studies of 131I-labeled mAb 14C5 and planar gamma imaging were done in nude mice bearing an A549 (non-small cell lung carcinoma) or a LoVo (colon carcinoma) tumor.
Internalization studies with both A549 and LoVo cells showed that 125I-labeled mAb 14C5 is slowly internalized with approximately 30% of the initially bound mAb 14C5 internalized after 2 hours at 37 degrees C. Internalization of mAb 14C5 could be visualized with confocal laser scanning microscopy. In vivo, radioisotope uptake peaked at 24 hours for both tumor models (n = 5) with no significant difference in percentage of injected dose/g tissue (A549 10.4 +/- 0.8 and LoVo 9.3 +/- 0.8). Via planar gamma camera imaging, A549 lung tumors as well as LoVo colon tumors could be clearly visualized.
The in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 are promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing antigen 14C5-expressing tumors.
