ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

Abstract

Purpose: To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy.

Methods: ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab.

Results: ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR.

Conclusion: (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Fig. 1

Purification and characterization of ALT-836-Fab. a Elution profile of ALT-836-Fab from a Sephadex G-75 column (arrowhead: the single fraction used for further in vitro and in vivo studies. b SDS-PAGE of ALT-836 (left lane) and ALT-836-Fab (right lane).

Fig. 2

Flow cytometry analysis of ALT-836-Fab at different concentrations (1 μg/mL and 5 μg/ml) in MDA-MB-231 (TF positive) and MDA-MB-435 (TF negative) cells. All data are repeated 3 times per group.

Fig. 3

Serial coronal PET images at different time points post-injection of ⁶⁴Cu-NOTA-ALT-836-Fab were acquired in MDA-MB-231 tumor-bearing mice, MDA-MB-231 tumor-bearing mice pre-injected with 2 mg ALT-836 (blocking), and MDA-MB-435 tumor-bearing mice.

Fig. 4

Quantitative analysis of the PET data. a Time activity curves of the liver, MDA-MB-231 tumor, blood, and muscle upon intravenous injection of ⁶⁴Cu-NOTA-ALT-836-Fab (positive group). b Time activity curves of the liver, MDA-MB-231 tumor, blood, and muscle upon intravenous injection of ⁶⁴Cu-NOTA-ALT-836-Fab after pre-injection of 2 mg ALT-836 (blocking group). c Time activity curves of the liver, MDA-MB-435 tumor, blood, and muscle upon intravenous injection of ⁶⁴Cu-NOTA-ALT-836-Fab (negative group). d Comparison of tumor/muscle ratio in positive, blocking and negative groups. The differences of the tumor uptake and tumor/muscle ratio in three groups were statistically significant (P < 0.05) at all time points except 0.5 h. All data represent 4 mice per group.

Fig. 5

Biodistribution of ⁶⁴Cu-NOTA-ALT-836-Fab in MDA-MB-231 tumor-bearing mice (positive group), MDA-MB-231 tumor-bearing mice pre-injected with 2 mg ALT-836 (blocking group), and MDA-MB-435 tumor-bearing mice (negative group) at 24 h post-injection (n = 4).

Fig. 6

Immunofluorescence TF/CD31 double-staining of MDA-MB-231 tumor and MDA-MB-435 tumor. Scale bar: 100 μm.

Fig. 7

Quantitative real time RT-PCR of TF expression in MDA-MB-231 and MDA-MB-435 tumors. a Agarose gel electrophoresis and b Critical time of RT-PCR products proved that TF has higher expression in the MDA-MB-231 tumor than the MDA-MB-435 tumor.