An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors

Abstract

Purpose: Prostate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies.

Methods: Yeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors.

Results: The A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo.

Conclusion: Of the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study.

Fig. 1

Parental hu1G8 (P) and affinity variant minibodies A2, A11, and C5. a Sequence of CDRs affected by residue substitution(s). b Schematic presentations of minibodies and scFv fragments. *Random residue substitution, V _L variable light, V _H variable heavy, C _H constant heavy

Fig. 2

Biological characterization. a SDS-PAGE analysis in non-reducing conditions of the four minibodies purified by protein L chromatography: parental (P), A11, A2, and C5. lane-M, molecular weight marker. b Size exclusion chromatography profile of A11 minibody purified by protein L chromatography on calibrated Superdex 200 Column. c A11 minibody stability; 1 mg/ml stock solution was stored at −20°C in 20% glycerol, or incubated at 4°C in PBS or 37°C in PBS/5% FBS for 5 days. Binding activity was determined by ELISA. Each dilution from 3,200 to 12,800 was assayed in triplicate

Fig. 3

Affinity ranking. a By competitive ELISA binding assay. Plates were coated with PSCA-mγ2a recombinant protein and biotinylated parental minibody was used as probe and mixed with different concentrations of non-labeled competitor: intact monoclonal antibody 1G8, minibody: parental (P), A2, A11, and C5. b By flow cytometry 50 nM of each minibody was incubated with PSCA-expressing cells. Cells were then stained with anti-human Fc PE-conjugated antibody. Negative control, A2, A11, C5, and P minibody as are indicated

Fig. 4

In vivo evaluation in SCID mice bearing LAPC-9 tumors that were injected with ¹²⁴I-labeled minibodies (A2, A11, and C5). a Coregistered microPET/microCT scan of SCID mice bearing LAPC-9 (PSCA-positive human prostate cancer) xenografts. The mice were injected with radiolabeled minibody variants (A2, A11, and C5) and scanned at 21 h post-injection. Coronal projections are presented that are adjusted to the same scale. b Biodistribution, Tu/Bl (tumor/blood) and imaging data Tu/Ba (tumor/background) at 21 h post-injection. Tumor and normal organ uptakes are expressed as percent injected dose per gram (%ID/g ± SE)

Fig. 5

Labeling of the parental and A11 minibodies. Labeling efficiency and immunoreactivity were determined for each minibody (n = 4). *The difference between the parental (P) (grey bars) and A11 (black bars) minibodies immunoreactivity was significant (p < 0.05)

Fig. 6

In vivo evaluation in nude mice bearing Capan-1 xenografts. a Coregistered microPET/microCT scan of ten nude mice bearing Capan-1 (PSCA-positive human pancreatic cancer) xenografts. The mice (n = 5) were injected with ¹²⁴I-radiolabeled minibody variants: parental (P) minibody top row, A11 minibody bottom row. Mice were scanned and biodistribution performed at 21 h post-injection. Coronal projections are presented that are adjusted to the same scale. b Biodistribution (n = 5) and Tu/Bl (tumor/blood). Tumor and normal organ uptakes are expressed as percent injected dose per gram (%ID/g ± SE)