Targeting prostate cancer cells in vivo using a rapidly internalizing novel human single-chain antibody fragment

Abstract

Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma.

Methods: A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with (99m)Tc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37 degrees C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection.

Results: The UA20 scFv was labeled in 55%-65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37 degrees C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 scFv. Kidney uptake was nonspecific, consistent with the route of excretion by scFvs.

Conclusion: The UA20 scFv showed rapid and specific internalization in prostate tumor cells in vitro and accumulation in prostate tumor xenografts in vivo, demonstrating the potential for future development for prostate cancer imaging and targeted therapy.

FIGURE 1

Cell binding and internalization of ^99mTc-labeled UA20. (A) Binding curves of ^99mTc-labeled UA20. Varying concentrations of antibodies were incubated with prostate cancer cell line DU145 at 4°C for 1 h, and number of cell-associated antibody molecules is plotted. (B) Internalization of ^99mTc-labeled UA20 after 1 h of incubation with prostate cancer cell line DU145 at 37°C. Percentage internalization is calculated from internalized amount over total bound amount.

FIGURE 2

Specificity of scFv internalization. Ten-fold excess of cold UA20 scFv was mixed with ^99mTc-labeled UA20 scFv and incubated with DU145 cells at 37°C for 1 h.

FIGURE 3

Tumor-to-nontumor ratio of ^99mTc-labeled UA20 in prostate cancer xenograft mice at 1 (◻) and 3 h (∎) after injection. Ratio is determined by dividing tumor uptake (%ID/g) by non-tumor uptake in each individual mouse and averaging (n = 4 in each group).

FIGURE 4

Small-animal SPECT/CT of nude mice bearing DU145 xenograft at front flank. (A) Image taken at 3 h after injection of ^99mTc-UA20 scFv. (B) Image taken at 3 h after injection of ^99mTc-labeled UA20 scFv. In this experiment, 10-fold excess of unlabeled UA20 scFv was injected 1 h before injection of ^99mTc-UA20 scFv. (C) Representative image of 3-dimensional rendering of tumor targeting by ^99mTc-labeled UA20 scFv.