Effect of antigen turnover rate and expression level on antibody penetration into tumor spheroids

Abstract

Poor tissue penetration is a significant obstacle to the development of successful antibody drugs for immunotherapy of solid tumors, and diverse alterations to the properties of antibody drugs have been made to improve penetration and homogeneity of exposure. However, in addition to properties of the antibody drug, mathematical models of antibody transport predict that the antigen expression level and turnover rate significantly influence penetration. As intrinsic antigen properties are likely to be difficult to modify, they may set inherent limits to penetration. Accordingly, in this study, we assess their contribution by evaluating the distance to which antibodies penetrate spheroids when these antigen properties are systematically varied. Additionally, the penetration profiles of antibodies against carcinoembryonic antigen and A33, two targets of clinical interest, are compared. The results agree well with the quantitative predictions of the model and show that localizing antibody to distal regions of tumors is best achieved by selecting slowly internalized targets that are not expressed above the level necessary for recruiting a toxic dose of therapeutic. Each antibody-bound antigen molecule that is turned over or present in excess incurs a real cost in terms of penetration depth-a limiting factor in the development of effective therapies for treating solid tumors.

Figure 1

Processing of spheroid images. A, original confocal images were transferred into ImageJ. B, they were then processed to eliminate background signal and generate binary data. C, a circular region of interest was drawn around each spheroid, and a readout of pixel intensity along a bisecting diagonal line was taken as the image was rotated in 18 projections around 360°. The number ofpixels with signal from each projection was then averaged, yielding the diameter of the spheroid that had been penetrated by label.

Figure 2

Antigen density affects spheroid penetration. A, LS174T spheroids were labeled with 1.5 nmol/L fluorescent A33 antibody (black) or 0.15 nmol/L fluorescent and 1.35 nmol/L nonfluorescent competitor (gray). The penetration distance ofthe fluorescent antibody into spheroids under each condition was highly correlated over time. B and C, representative image of an LS174T spheroid at 24 h labeled with 0.15 nmol/L fluorescent and 1.35 nmol/L nonfluorescent competitor (B) or 1.5 nmol/L fluorescent antibody (C). D, ratio of penetration for spheroids with differing numbers of available binding sites (:10) at 0.15, 0.7, and 1.5 nmol/L doses of fluorescent antibody at 24 and 48 h. The penetration ratio is a maximum of 3.2 at the lowest concentration, very close to the predicted value of 10 (horizontal line). Over time and at greater concentrations, this ratio approaches a value of 1 as the spheroids become saturated, setting an upper limit on the penetration distance.

Figure 3

Antigen density affects spheroid penetration. A and B, representative images of an LS174T (A) and SW1222 (B) spheroids labeled with 1 nmol/L A33 antibody at 12 h. C, penetration distance into SW1222 (black) and LS174T (gray) spheroids at 12 h. D, ratio of penetration (SW1222/LS174T) at 12 (black) and 24 (gray) h and the predicted value of 2.45 (horizontal line).

Figure 4

Antigen internalization and turnover affects penetration. A and B, representative images ofLS174T spheroids labeled with 1.5 nmol/L CEA antibody M85151a (A) or M111147 (B). C, ratio of penetration depth (slow M111147/fast M85151a) over a range of concentrations at 48 h compared with the predicted value of 2.3. At greater concentrations, this ratio damps out to a value of 1 as the spheroids become saturated, setting an upper limit on the penetration distance.

Figure 5

Antigen internalization reaches a steady-state with diffusion and can limit penetration. Penetration of anti-CEA (A) and anti-A33 (B) antibodies into LS174T spheroids. At low concentrations, anti-CEA antibody penetration plateaus at a given radius, whereas antibodies to A33 accomplish penetration at much lower concentrations. C, representative images of spheroids at 24 (left) and 48 (right) h with 1.5 nmol/L anti-CEA antibody (top), 1.5 nmol/L anti-A33 antibody (middle), and 0.07 nmol/L anti-A33 antibody. After 24 h, antibody to CEA does not penetrate further into the spheroid mass, despite elapsed time.

Figure 6

Differential in-spheroid turnover and accessibility of A33 and CEA antibody over time. A, images of LS174T spheroids grown in A33 (left) or CEA (right) antibody. Antibody was then removed from the bulk, and spheroids were imaged 1 to 4 d postremoval to follow the localization and persistence offluorescent antibody. B, 4 d after label was washed out, spheroids were labeled with an antimouse antibody conjugated with PE, allowing identification of the surface accessible primary antibody.