PET imaging of CD105/endoglin expression with a ⁶¹/⁶⁴Cu-labeled Fab antibody fragment.
OBJECTIVE
The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e., endoglin), and investigate its potential for PET imaging of tumor angiogenesis in a small-animal model after (61/64)Cu labeling.


METHODS
TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and (61/64)Cu labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficiency and normal organ distribution of (61/64)Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression.


RESULTS
TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu labeling of NOTA-TRC105-Fab was achieved with about 50 % yield (specific activity about 44 GBq/μmol). PET imaging revealed rapid uptake of (64)Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6 ± 0.4, 4.2 ± 0.5, 4.9 ± 0.3, 4.4 ± 0.7, and 4.6 ± 0.8 %ID/g at 0.5, 2, 5, 16, and 24 h after injection, respectively; n = 4). Since tumor uptake peaked soon after tracer injection, (61)Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h after injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination.


CONCLUSIONS
We report PET imaging of CD105 expression using (61/64)Cu-NOTA-TRC105-Fab, which exhibited prominent and target-specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to radiolabeled intact antibody, which may be translated into same-day immunoPET imaging for clinical investigation.
Department of Medical Physics, University of Wisconsin - Madison, 1111 Highland Avenue, Madison, WI 53705-2275, USA.
Yin
Y
Zhang
Hao
H
Hong
Hakan
H
Orbay
Hector F
HF
Valdovinos
Tapas R
TR
Nayak
Charles P
CP
Theuer
Todd E
TE
Barnhart
Weibo
W
Cai
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD / Q000378:metabolism*; D003301:Copper Radioisotopes; D000071063:Endoglin; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007553:Isotope Labeling; D061986:MCF-7 Cells; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply / Q000000981:diagnostic imaging; D051379:Mice; D009389:Neovascularization, Pathologic; D049268:Positron-Emission Tomography / Q000379:methods*; D011874:Radiometry; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D003301:Copper Radioisotopes; C064206:ENG protein, human; D000071063:Endoglin; D007140:Immunoglobulin Fab Fragments; D011956:Receptors, Cell Surface; C579557:carotuximab