99mTc-labelled S-HYNIC certolizumab pegol in rheumatoid arthritis and spondyloarthritis patients: a biodistribution and dosimetry study.

BACKGROUND: Biologicals directed against tumour necrosis factor (TNF) have proven their efficacy in the treatment of spondyloarthritis and rheumatoid arthritis. We present a radiolabelling method for certolizumab pegol (CZP), a commercially available humanized Fab'-fragment directed against TNF. A biodistribution and dosimetry study was conducted. Tc-S-HYNIC CZP was synthesized. The in vitro TNF neutralizing activity was tested by exposing L929s-cells to various concentrations 99mTc-S-HYNIC CZP and measuring TNF-induced cytotoxicity. For biodistribution and dosimetry, WB images and blood and urine sampling were performed up to 24 h pi. Cumulative activities were estimated using mono-exponential fitting, and organ doses were estimated using OLINDA/EXM. The effective dose was calculated using the International Commission on Radiological Protection 103 recommendations. The uptake of the tracer in the peripheral joints was assessed visually and semiquantitatively. RESULTS: In vitro tests showed blocking of TNF cytotoxicity by the 99mTc-S-HYNIC CZP formulation comparable to the effect obtained with the unlabelled CZP with or without the HYNIC linker. We analysed eight patients with rheumatoid arthritis or spondyloarthritis. The highest mean absorbed organ doses were recorded for kidneys, spleen, and liver: 56 (SD 7), 34 (SD 6), and 33 (SD 7) µGy/MBq. The effective dose was 6.1 (SD 0.9) mSv for a mean injected activity of 690 (SD 35) MBq. The urinary excretion was 15.1% (SD 8.1) of the IA at 22.5 h. Blood analysis yielded a distribution half-life of 1.2 h (SD 1.5) and an elimination half-life of 26.9 h (SD 2.7). Visual analysis of the scans revealed marked tracer accumulation in the clinically affected peripheral joints. In addition, there was a statistically significant higher uptake of the tracer in the swollen joints (median uptake ratio compared to background of 3.3 in rheumatoid arthritis and 2.4 in peripheral spondyloarthritis) compared to clinically negative joints (respectively 1.3 and 1.6). CONCLUSIONS: We present a radiolabelling technique for CZP, a Fab'-fragment directed against TNF and currently used as a therapeutic agent in rheumatology. An effective dose of 6.1 mSv (SD 0.9) was estimated. We confirmed the uptake of this new radiopharmaceutical in clinically affected peripheral joints.

Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function.

INTRODUCTION: Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug-drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [(99m)Tc]-DTPA-CDCA and [(99m)]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality. METHODS: [99mTc]-DTPA-CDCA([(99m)Tc]-3a) and [99mTc]-DTPA-CA ([(99m)Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. Km and Vmax values were determined and compared to [(99m)Tc]-mebrofenin ([(99m)Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n=3) and rifampicin treated (n=3) mice were intravenously injected with 37MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of heart, liver, gallbladder and intestines were obtained. RESULTS: We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [(99m)Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [(99m)Tc]-3a shows higher affinities compared to [(99m)Tc]-3b. Vmax values were lower compared to [(99m)Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin. CONCLUSION: The involved transporters were identified; both tracers are taken up in the hepatocytes by OATP1B1 andOATP1B3 with Km and Vmax values in the same range as endogenous bile acids and are secreted into bile canaliculi via MRP2. Dynamic small-animal SPECT imaging can be a useful noninvasive method of visualizing and quantifying hepatobiliary transporter functionality and disturbances thereof in vivo, which could predict drug pharmacokinetics.

Generation and in vivo characterization of a chimeric αvß5-targeting antibody 14C5 and its derivatives.

BACKGROUND: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvß5 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 αvß5 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.

In vivo visualization and quantification of (Disturbed) Oatp-mediated hepatic uptake and Mrp2-mediated biliary excretion of 99mTc-mebrofenin in mice.

UNLABELLED: Hepatic transport of (99m)Tc-mebrofenin through organic anion transport protein 1a and 1b (Oatp1a/1b) and multidrug resistance protein 2 (Mrp2) was investigated by small-animal SPECT. On the basis of the results, a noninvasive method to visualize and quantify disturbances in hepatic transport is proposed. METHODS: Friend virus B wild-type mice (untreated, bile duct-ligated, vehicle- or rifampicin-treated) and strain-matched knockout mice unable to express the uptake transporters Oatp1a/1b (Slco1a/1b(-/-)/(-/-)) or the efflux transporter Mrp2 (Abcc2(-/-)) were intravenously injected with (99m)Tc-mebrofenin (n = 3 per group). After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of the liver and of the gallbladder and intestines were obtained and correlated with direct blood samples. RESULTS: Normal hepatobiliary clearance of (99m)Tc-mebrofenin was severely impaired in the bile duct-ligated animal, as evidenced by elevated hepatic tracer levels. In Slco1a/1b(-/-)/(-/-) mice, a lower area under the curve (AUC) for the liver (P = 0.014) was obtained and no activity was detected in the gallbladder and intestines. Renal rerouting was observed, along with an increase in the blood AUC (P = 0.01). Abcc2(-/-) mice had a higher liver AUC (P = 0.009), a delayed emergence time of (99m)Tc-mebrofenin in the gallbladder (P = 0.009), and a lower AUC for the gallbladder and intestines (P = 0.001). The blood curve was similar to that of wild-type mice. (99m)Tc-mebrofenin disposition was altered after rifampicin treatments. We observed a dose-dependent delayed time point at which tracer maximized in liver, an increased AUC for liver, and a lower AUC for gallbladder and intestines (P = 0.042, 0.034, and 0.001, respectively, highest dose). Emergence in the gallbladder occurred later (P = 0.009, highest dose), and blood AUC was higher (P = 0.006). CONCLUSION: The current study visualized and quantified hepatic uptake and biliary efflux of (99m)Tc-mebrofenin. Our results demonstrated the possibility of discriminating, on a quantitative level, between lack of functional activity of sinusoidal uptake versus that of biliary efflux transporters.

The influence of mass of [11C]-laniquidar and [11C]-N-desmethyl-loperamide on P-glycoprotein blockage at the blood-brain barrier.

INTRODUCTION: An earlier report suggested that mass amount of PET tracers could be an important factor in brain uptake mediated by P-glycoprotein. Thereby, this study investigated the influence of mass dose of laniquidar, desmethyl-loperamide and loperamide on the P-glycoprotein-mediated brain uptake of, respectively, [(11)C]-laniquidar and [(11)C]-N-desmethyl-loperamide ([(11)C]-dLop). METHODS: Wild-type (WT) mice were injected intravenously with solutions of 5.6 MBq [(11)C]-laniquidar (either no carrier added or 60 mg/kg laniquidar added) or with 5.0-7.4 MBq [(11)C]-dLop (either no carrier added or 3 mg/kg desmethyl loperamide). Mice were killed, and brain and blood were collected, weighted and counted for radioactivity. Mdr1a(-/-) knockout mice were incorporated as the control group. RESULTS: Injection of (11)C-laniquidar (no carrier added) in WT mice resulted in a statistical significant lower brain uptake (0.7±0.2 %ID/g) compared to the carrier-added formulation (60 mg/kg laniquidar) (3.1±0.3 %ID/g) (P=.004), while no statistical difference could be observed between formulations of [(11)C]-dLop. The [(11)C]-laniquidar and [(11)C]-dLop blood concentrations were not significantly different between the tested formulations in WT mice. In control animals, no effect of mass amount on brain uptake of both tracers could be demonstrated. CONCLUSIONS: These results demonstrate the bivalent character of laniquidar, acting as a substrate at low doses and as a blocking agent for P-glycoprotein transport in the brain at higher doses. In comparison, no difference was observed in [(11)C]-dLop uptake between carrier- and no-carrier-added formulations, which confirms that desmethyl-loperamide is a substrate of P-glycoprotein at the blood-brain barrier.

Reduced dimethylaminoethanol in [(18)F]fluoromethylcholine: an important step towards enhanced tumour visualization.

PURPOSE: [(18)F]Fluoromethylcholine ([(18)F]FCho) is a radiotracer generally used for tumour visualization in patients. Due to high levels of dimethylaminoethanol (DMAE) remaining in [(18)F]FCho solutions synthesized by currently available methods, tumour visualization might be compromised. METHODS: An improved purification method involving an optimized purification step for reducing the levels of DMAE was conceived. The physiological explanation for the interference of residual DMAE in [(18)F]FCho pharmacokinetics was further elaborated in a xenograft mouse model. RESULTS: The use of a series of polymer solid-phase extraction cartridges (Oasis HLB/WCX), instead of the commonly used combination of tC18 and Accell CM cartridges, reduced DMAE levels from 402.2±49.6 ppm to 3.0±0.5 ppm. Subsequent in vitro tests proved that (1) [(18)F]FCho uptake was reduced in the presence of DMAE at concentrations above 0.5 µM and (2) DMAE is a competitive inhibitor of [(18)F]FCho transport. In vivo experiments in xenograft mouse models corroborated reduced tumour uptake at DMAE plasma levels of about 2.5 µM as found in patients injected with contaminated [(18)F]FCho. CONCLUSION: Residual DMAE, even at levels below choline plasma concentrations found during fasting, compromises [(18)F]FCho uptake in vivo and care should be taken to avoid its interference in molecular imaging with [(18)F]FCho.

Preclinical evaluation of monoclonal antibody 14C5 for targeting pancreatic cancer.

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.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

BACKGROUND: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. RESULTS: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. CONCLUSION: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.

Monoclonal antibody 14C5 targets integrin alphavbeta5.

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.