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The human monoclonal antibody (MAb) fragment L19-SIP is directed against extra domain B (ED-B) of fibronectin, a marker of tumour angiogenesis. A clinical radioimmunotherapy (RIT) trial with 131I-L19-SIP was recently started. In the present study, after GMP production of 124I and efficient production of 124I-L19-SIP, we aimed to demonstrate the suitability of 124I-L19-SIP immuno-PET for imaging of angiogenesis at early-stage tumour development and as a scouting procedure prior to clinical 131I-L19-SIP RIT. 124I was produced in a GMP compliant way via 124Te(p,n)124I reaction and using a TERIMO™ module for radioiodine separation. L19-SIP was radioiodinated by using a modified version of the IODO-GEN method. The biodistribution of coinjected 124I- and 131I-L19-SIP was compared in FaDu xenograft-bearing nude mice, while 124I PET images were obtained from mice with tumours of <50 to ∼700 mm3. 124I was produced highly pure with an average yield of 15.4 ± 0.5 MBq/μAh, while separation yield was ∼90% efficient with <0.5% loss of TeO2. Overall labelling efficiency, radiochemical purity and immunoreactive fraction were for 124I-L19-SIP: ∼80 , 99.9 and >90%, respectively. Tumour uptake was 7.3 ± 2.1, 10.8 ± 1.5, 7.8 ± 1.4, 5.3 ± 0.6 and 3.1 ± 0.4%ID/g at 3, 6, 24, 48 and 72 h p.i., resulting in increased tumour to blood ratios ranging from 6.0 at 24 h to 45.9 at 72 h p.i.. Fully concordant labelling and biodistribution results were obtained with 124I- and 131I-L19-SIP. Immuno-PET with 124I-L19-SIP using a high-resolution research tomograph PET scanner revealed clear delineation of the tumours as small as 50 mm3 and no adverse uptake in other organs. 124I-MAb conjugates for clinical immuno-PET can be efficiently produced. Immuno-PET with 124I-L19-SIP appeared qualified for sensitive imaging of tumour neovasculature and for predicting 131I-L19-SIP biodistribution. [ABSTRACT FROM AUTHOR]

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Unlabelled: The extra domain B of fibronectin (ED-B) is a marker of tumor angiogenesis. The human monoclonal antibody (mAb) L19-SIP (approximately 80 kDa; SIP is "small immunoprotein") has been selected for targeting of ED-B. The aim of this study was to evaluate the potential of radioimmunotherapy (RIT) with L19-SIP, either alone or in combination with cetuximab, for treatment of head and neck squamous cell carcinoma (HNSCC). Combination with cetuximab was considered because this anti-EGFR (epidermal growth factor receptor) mAb has proven value for the treatment of HNSCC., Methods: HNSCC xenograft lines FaDu and HNX-OE were evaluated for ED-B and EGFR expression. L19-SIP was radiolabeled with 2 candidate radionuclides for RIT, 177Lu and 131I (or 125I as substitute). The biodistribution of coinjected 177Lu-L19-SIP and 125I-L19-SIP was assessed in FaDu-bearing nude mice, whereas 131I-L19-SIP was evaluated in both xenograft lines. After labeling with high-dose 131I (623-789 MBq/mg), the maximum tolerated dose (MTD) was assessed. The efficacy of RIT with injected 131I-L19-SIP, either alone or in combination with unlabeled cetuximab (1 mg 2 times a week intraperitoneally for 4 wk), was evaluated in both xenograft lines., Results: Xenograft lines expressed both antigens, with similar EGFR expression and the highest ED-B expression in FaDu. Radioiodinated L19-SIP performed better than 177Lu-L19-SIP and was further exploited. The biodistribution of 131I-L19-SIP was most favorable in FaDu-bearing mice, with tumor uptake values at 24, 48, and 72 h after injection of 8.6 +/- 1.6, 5.8 +/- 0.4, and 3.4 +/- 0.2 %ID/g (%ID/g is percentage injected dose per gram of tissue), respectively, and ratios of tumor to normal tissues that gradually increased in time, such as for blood from 4.4 +/- 1.8 at 24 h to 21.4 +/- 1.7 at 72 h, after injection. RIT at the MTD level of 74 MBq caused significant tumor growth delay and improved survival in both lines. Although FaDu was most sensitive for RIT, with size reduction of all tumors, HNX-OE was most sensitive for treatment with cetuximab. The best survival and cure rates were obtained, however, when RIT and cetuximab were combined., Conclusion: RIT with 131I-L19-SIP appeared efficacious in HNSCC xenografts. The efficacy of RIT was enhanced by combination with cetuximab, without increase of toxicity.