While few reports suggest FcRn may not contribute significantly toward tissue distribution of mAbs, 14 a recent report proposes that FcRn may play a significant role in mAb biodistribution.15 Our data (Fig.?5) shows that the relationship between protein size and tissue uptake holds true for both FcRn binding and non-FcRn binding antibody fragments, suggesting an insignificant contribution of FcRn in transcytosis-mediated tissue uptake. In summary, we demonstrated that the linear relationship between tissue and plasma concentrations can be generalized to different sizes of antibody fragments. in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35?kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. -?and of a hypothetical protein with molecular weight approaching zero, and the slope parameter “provides a value in kDa, representing the molecular weight increase that results in 50% reduction of the BC value (BC50) and tissue uptake. Estimates of Vitamin E Acetate these parameters for each tissue, as well as the R2 values of the fittings, are listed in Table?2. The model fittings are shown as the solid line in the Figure?4. The resultant equation well captured the data in all the tissues with good R2 values. No R2 value was reported for skin, brain, and pancreas because only 2 BCs were available for fitting these 3 tissues. Table 2. Estimated parameters for the BC vs. protein molecular weight relationship. -?and of a hypothetical protein with radius approaching zero, and the slope parameter “prediction of their absorption, distribution, Vitamin E Acetate metabolism, and excretion (ADME) properties. Such relationships enable rational (e.g.,, model based) drug development and minimize the dependence on animals to gain ADME information. Toward this goal, here we established the first ever continuous quantitative relationship between protein size and tissue distribution by deriving BC values for different antibody fragments using biodistribution data from numerous published studies. A general comparison of the extent of tissue distribution between mAb and the fragments (Table?1) reveal that the distribution of mAb fragments into the tissues is Rabbit Polyclonal to MLH1 higher compared to mAb. These findings are expected and consistent with the reports that suggest that tissue penetration of smaller proteins is relatively easier due to their greater extravasation through the pores of vascular capillary wall.2,3 Additionally, when the BC values were compared across the investigated proteins, several other general Vitamin E Acetate trends were observed. First, kidney represented the most significant distribution site for mAb fragments. BC values in kidney were estimated to be the highest for all 3 fragments (0.71 for F(ab’)2, 7.34 for Fab’ and 12.35 for scFv). For mAb, the BC value in kidney was much lower (0.14), and did not exceed above all other tissues.1 We hypothesize that this observation is probably due to renal filtration of smaller antibody fragments, followed by their extensive reabsorption.4 It is known that molecules with an effective radius of less than 1.8?nm (or 12?kDa) are freely filtered, and molecules with 4.2?nm (or 70?kDa) are ?ltered to a limited extent.5 However, even molecules larger than 70?kDa have been reported to accumulate in kidney to a limited extent.6-8 Another factor that could influence these high BC values for the kidney is the distribution of the free label to the kidney. However, since we find increasing BC values with decreasing size of antibody fragments irrespective of the labeling, this effect may not contribute significantly. Second, it was observed that clear differences in fitted BC values could be found between “tight” and “leaky” tissues. “Leaky” tissues, such as liver, lung and spleen had higher BC values than “tight” tissues like muscle, heart and brain. It was also observed that scFv demonstrated very high distribution into the gastro-intestinal (GI) tract. In our Vitamin E Acetate knowledge there is no published information available to explain this phenomenon, but one hypothesis could be that this is related to a hepatobiliary elimination process. Lastly, compared to mAb BC values, the BC value of F(ab’)2 in brain showed the greatest (more than 10-fold) increase. This could either be related to the reduced protein size or possibly due to the absence of the Fc fragment, which has been hypothesized to contribute to mAb efflux from the brain.9,10 It should be.