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Differentiation between temporary and real non-clearability of biotinylated IgG antibody by avidin in mice.

Dou S, Virostko J, Rusckowski M, Greiner DL, Powers AC, Liu G - Front Pharmacol (2014)

Bottom Line: By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective.By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays.In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, University of Massachusetts Medical School Worcester, MA, USA.

ABSTRACT
Although an increasing number of antibody conjugates are being used in the clinic, there remain many unmet needs in antibody targeting. Normal tissue background is one of the key issues that limits the therapeutic efficacy and the detection sensitivity. Background reduction coupled with dose increase may provide the required target accumulation of the label or toxin at an acceptable normal tissue background. However, the knowledge about the in vivo interaction between antibody and a clearing agent is currently inadequate for designing a rational clearance regimen or system. The current investigation focuses on the clearability of antibody for background reduction, an important topic to antibody targeting in general. The investigation employs pretargeting as a research tool and avidin as a model clearing agent. By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective. By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays. The study of multiple avidin injections confirmed that the presence of clearable biotinylated antibodies after an avidin injection is due to their temporary inaccessibility and subsequent return from tissue compartments. The collective clearance efficiency of 91% by three avidin injections indicates a continuous IV infusion would be recommended to remove all of the biotinylated IgG molecules. In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.

No MeSH data available.


The blood radioactivity levels after the injection of the biotin-Ab-MORF, different numbers of injections of avidin, and the injection of the radiolabeled cMORF. The time of euthanization was at 4 days after initial injection of the pretargeting antibody and 3 h after injection of radiolabeled cMORF (see Materials and Methods).
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Figure 3: The blood radioactivity levels after the injection of the biotin-Ab-MORF, different numbers of injections of avidin, and the injection of the radiolabeled cMORF. The time of euthanization was at 4 days after initial injection of the pretargeting antibody and 3 h after injection of radiolabeled cMORF (see Materials and Methods).

Mentions: As shown in Figure 3, additional injections of avidin subsequent to the first further reduce the blood antibody concentration. This indicates that the majority of antibody molecules that survived the first avidin injection are clearable. The collective clearance efficiency following 3 avidin injections is 80 ± 2% (0.61/3.10*100%). The separate clearance efficiency following each injection is 43 ± 12% (1 − 1.78/3.10), 44 ± 11% (1 − 0.99/1.78), and 38 ± 7% (1 − 0.61/0.99), respectively. The real clearance efficiency may actually be higher because of the non-clearable portion. A calculation based on a constant clearance efficiency of 51% (higher than 38–44%), and the non-clearable portion of 12% provides the blood concentrations of 3.10, 1.71, 1.03, 0.69%ID/g. These numbers are in a good agreement to the measured 3.10 ± 0.24, 1.78 ± 0.33, 0.99 ± 0.07, 0.61 ± 0.05%ID/g.


Differentiation between temporary and real non-clearability of biotinylated IgG antibody by avidin in mice.

Dou S, Virostko J, Rusckowski M, Greiner DL, Powers AC, Liu G - Front Pharmacol (2014)

The blood radioactivity levels after the injection of the biotin-Ab-MORF, different numbers of injections of avidin, and the injection of the radiolabeled cMORF. The time of euthanization was at 4 days after initial injection of the pretargeting antibody and 3 h after injection of radiolabeled cMORF (see Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109569&req=5

Figure 3: The blood radioactivity levels after the injection of the biotin-Ab-MORF, different numbers of injections of avidin, and the injection of the radiolabeled cMORF. The time of euthanization was at 4 days after initial injection of the pretargeting antibody and 3 h after injection of radiolabeled cMORF (see Materials and Methods).
Mentions: As shown in Figure 3, additional injections of avidin subsequent to the first further reduce the blood antibody concentration. This indicates that the majority of antibody molecules that survived the first avidin injection are clearable. The collective clearance efficiency following 3 avidin injections is 80 ± 2% (0.61/3.10*100%). The separate clearance efficiency following each injection is 43 ± 12% (1 − 1.78/3.10), 44 ± 11% (1 − 0.99/1.78), and 38 ± 7% (1 − 0.61/0.99), respectively. The real clearance efficiency may actually be higher because of the non-clearable portion. A calculation based on a constant clearance efficiency of 51% (higher than 38–44%), and the non-clearable portion of 12% provides the blood concentrations of 3.10, 1.71, 1.03, 0.69%ID/g. These numbers are in a good agreement to the measured 3.10 ± 0.24, 1.78 ± 0.33, 0.99 ± 0.07, 0.61 ± 0.05%ID/g.

Bottom Line: By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective.By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays.In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology, University of Massachusetts Medical School Worcester, MA, USA.

ABSTRACT
Although an increasing number of antibody conjugates are being used in the clinic, there remain many unmet needs in antibody targeting. Normal tissue background is one of the key issues that limits the therapeutic efficacy and the detection sensitivity. Background reduction coupled with dose increase may provide the required target accumulation of the label or toxin at an acceptable normal tissue background. However, the knowledge about the in vivo interaction between antibody and a clearing agent is currently inadequate for designing a rational clearance regimen or system. The current investigation focuses on the clearability of antibody for background reduction, an important topic to antibody targeting in general. The investigation employs pretargeting as a research tool and avidin as a model clearing agent. By comparing the effects of natural clearance at a longer post-injection time and avidin clearance, we demonstrated that avidin clearance is much more effective. By directly attaching avidin to a biotinylated antibody prior to injection, we found that the biotinylated antibody in blood, once bound to the clearing agent, can be removed from the circulation immediately and completely, while the real non-clearable antibody without biotin stays. The study of multiple avidin injections confirmed that the presence of clearable biotinylated antibodies after an avidin injection is due to their temporary inaccessibility and subsequent return from tissue compartments. The collective clearance efficiency of 91% by three avidin injections indicates a continuous IV infusion would be recommended to remove all of the biotinylated IgG molecules. In conclusion, the use of antibody pretargeting as a tool in this study has improved understanding of the incomplete clearance by avidin and can aid in overcoming this obstacle.

No MeSH data available.