Annemieke C. Soede

Specific imaging of VEGF-A expression with radiolabeled anti-VEGF monoclonal antibody.

Vascular endothelial growth factor‐A (VEGF‐A) is one of the most important angiogenic factors. Here, we studied in a nude mouse model whether the expression of VEGF‐A in a tumor could be imaged with a radiolabeled anti‐VEGF antibody. The humanized anti‐VEGF‐A antibody A.4.6.1. (bevacizumab), which is reactive with all VEGF‐A isoforms, was radiolabeled with In‐111 or with I‐125. The accumulation of the radiolabeled antibodies in VEGF‐A expressing tumors (LS174T) in nude mice was examined in biodistribution studies and by gamma camera imaging. The uptake of the In‐111‐bevacizumab in the tumor at 3 days p.i. was significantly higher than that of I‐125‐bevacizumab (19.4 ± 7.0 %ID/g vs. 9.6 ± 3.3 %ID/g, p = 0.04). Coinjection of an excess unlabeled antibody resulted in a significant decrease in radioactivity concentration in the tumor (<2.9 ± 1.9 %ID/g, p < 0.005), indicating VEGF‐mediated antibody uptake. Highest uptake in the tumor was observed at relatively low antibody protein doses (<3 μg) (20–25 %ID/g). VEGF‐A‐expressing tumors could be clearly visualized on planar scintigraphic images from 24‐hr post injection onwards. In conclusion, VEGF‐A expression in tumors can be visualized specifically with radiolabeled anti‐VEGF‐A‐mAb.

Experimental radioimmunotherapy of small peritoneal metastases of colorectal origin

Radioimmunotherapy using radiolabeled monoclonal antibodies (MoAbs) directed against tumor‐associated antigens might be an effective treatment modality for small volume disease. Our aim was to optimize an experimental model of radioimmunotherapy for small peritoneal metastases of colorectal origin using the anti‐CEA MoAb MN‐14. In nude mice with intraperitoneal (i.p.) LS174T tumors, a protein dose‐escalation study was carried out to determine the maximal dose of radioiodinated MN‐14 to be used in radioimmunotherapy. The biodistribution of radioiodinated MN‐14 was determined after intravenous (i.v.) and i.p. administration. Finally, the therapeutic efficacy of escalating activity doses of 131I‐labeled MN‐14 (62.5–500 μCi) was assessed and compared to that of unlabeled MN‐14 or 500 μCi of 131I‐labeled irrelevant control antibody. At protein doses higher than 25 μg, uptake in tumor was reduced, presumably due to saturation of tumor antigen. During the first 24 hours i.p. administration led to higher tumor uptake and higher tumor:blood ratios than i.v. administration. Median survival of the control groups was 38 days (unlabeled MN‐14) and 52 days (131I‐labeled nonspecific antibody). Median survival of the groups treated with increasing activity doses of 131I‐labeled MN‐14 was 42 days (62.5 μCi), 49 days (125 μCi), 63 days (250 μCi) and 101 days (500 μCi), respectively (p < 0.0001 compared to unlabeled MN‐14). The present study shows that the anti‐CEA‐antibody MN‐14 preferentially accumulates in i.p. LS174T tumor xenografts after both i.p. and i.v. administration. Intraperitoneal radioimmunotherapy using 131I‐labeled MN‐14 delays significantly the outgrowth of peritoneal LS174T metastases, even at relatively low activity doses.

Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles

This study focused on the degradation properties of gelatin microparticles incorporated in calcium phosphate (CaP) cement and the subsequent effect of these composites on bone formation. Positively charged alkaline gelatin (type A) microparticles or negatively charged acidic gelatin (type B) microparticles were incorporated in CaP cement, which was implanted in critical-sized cranial defect in rats and left in place for 2, 4, and 8 weeks. The degradation of the gelatin was monitored using radioiodinated microparticles. After 4 and 8 weeks of implantation, a significantly faster degradation of type A gelatin over type B gelatin was found. Light microscopic analysis of the specimens showed similar bone response concerning implants containing either type A or B gelatin microparticles. At 2 weeks of implantation, a minimal amount of bone formation was observed from the cranial bone toward the implant, while after 8 weeks of implantation an entire layer of newly formed bone was present from the cranial bone toward the implant periphery. Bone ingrowth into the implant was observed at sites of gelatin microparticle degradation, predominantly at the implant periphery. Histomorphometrical evaluation did not reveal significant differences in bone formation between CaP cement incorporated with either type A or B gelatin microparticles during implantation periods up to 8 weeks. In conclusion, this study demonstrates that gelatin type influences the degradation of gelatin microparticles incorporated in CaP cements. However, this difference in degradation and the concomitant subsequent macroporosity did not induce differences in the biological response.

Pretargeting of carcinoembryonic antigen-expressing tumors with a biologically produced bispecific anticarcinoembryonic antigen x anti-indium-labeled diethylenetriaminepentaacetic acid antibody.

Purpose: The aim of these studies was to develop a pretargeting strategy for CEA-expressing cancers using biologically produced bispecific monoclonal antibodies (bsMAb). The bsMAbs used in this system have affinity for the carcinoembryonic antigen on the one hand, and for indium-labeled diethylenetriaminepentaacetic acid (DTPA), on the other. Experimental Design: Stable quadroma clones producing bsMAb MN-14xDTIn-1 were isolated. LS174T tumor–bearing mice were injected with 1 to 100 μg of bsMAb followed by 1 to 60 ng of an 111In-labeled bivalent peptide [Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2]. Mice were killed at 24 hours postinjection and the biodistribution of the radiolabel was determined. The biodistribution of diDTPA labeled with four different radionuclides (111In, 99mTc, nonresidualizing 125I, and residualizing 125I) was determined at various time points postinjection following pretargeting of LS174T tumors with bsMAb MN-14xDTIn-1. Results: Optimal tumor targeting was observed when tumors were pretargeted with 10 μg of bsMAb MN-14xDTIn-1 and when 6 ng of a radiolabeled peptide was given 72 hours later. The uptake of the four radiolabels in LS174T tumors at 4 hours postinjection was similar. However, at later time points, the 111In-label and residualizing 125I-label were better retained in the tumor than the nonresidualizing 125I label. Although the absolute uptake in the tumor (in terms of percentage of injected dose per gram of tissue) was 5-fold lower than the uptake obtained with directly labeled MN-14, the pretargeting strategy revealed much higher tumor-to-blood ratios due to the rapid clearance of the radiolabel from the circulation as compared with 111In-MN-14 (445 ± 90 and 5.3 ± 1.1, respectively, at 72 hours postinjection). Conclusions: Effective targeting of carcinoembryonic antigen-expressing tumors was achieved with a newly produced bispecific antibody. The 111In-labeled l-amino acid peptide and 125I-d-amino acid peptide were better retained in the tumor than the 99mTc- and 125I-l-amino acid peptide. Very high tumor-to-blood ratios were obtained due to rapid background clearance.

Comparison of IgG and F(ab')2 fragments of bispecific anti-RCCxanti-DTIn-1 antibody for pretargeting purposes.

PurposeAn effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCC×anti-DTPA(In) (bsMAb: G250×DTIn-1). Tumour uptake of a 111In-labelled bivalent peptide after pretargeting with bsMAb G250×DTIn-1 was relatively high compared with that in other pretargeting systems using chemically coupled F(ab′)2 fragments. Here, we investigated the effect of the bsMAb form in the pretargeting strategy.MethodsTo determine the optimal interval between the administration of each of the bsMAb forms and the 111In-labelled bivalent peptide, the biodistribution of the radioiodinated bsMAb forms was studied in athymic mice with subcutaneous SK-RC-1 RCC tumours. Since tumour targeting of the radiolabelled peptide depends on the bsMAb form and dose, a bsMAb dose escalation study was carried out for both bsMAb forms. Under optimised conditions, the biodistribution of the 111In label in mice with pretargeted RCC was determined from 4xa0h up to 7 days p.i.ResultsThe optimal interval between the two administrations was 72xa0h for the bsMAb IgG and 4xa0h for the bsMAb F(ab′)2. The optimal bsMAb dose for intact IgG was 67xa0pmol and the optimal bsMAb F(ab′)2 dose was 200xa0pmol. Targeting of the pretargeted RCC with 4xa0pmol 111In-labelled bivalent peptide revealed high tumour uptake with both bsMAb forms.ConclusionWith the pretargeting strategy, using either bsMAb IgG or bsMAb F(ab′)2, very efficient peptide targeting of the tumour was obtained. Uptake and retention of the radiolabel in the tumour with the pretargeting approach are not affected by the bsMAb form used.